Public Transportation in Puglia

Brindisi → Ostuni
Easy transfer. STP Brindisi Bus or FlixBus
Ostuni → Matera
More complicated. Probably the best is to take a train to Bari then go to Matera via train or bus (bus better and faster)
Matera → Alberobello
Very easy by car (it was just over 1h drive for me last year), but time consuming by public transit as you need a bus or train (bus better) to Bari, then a bus to Alberobello (very frequent and fast)
Alberobello → Bari
Easy bus transfer, very frequent. See above

Assuming you want to fly to Brindisi (I'd fly to Bari for your plan), it is better to change your itinerary as follows:
Brindisi - Ostuni - Alberobello - Bari - Matera

If you fly to Bari (preferable in my opinion):
Bari - Matera - Alberobello (via Bari) - Ostuni - Bari.

If you can rent a car, that would be great. This year and last year I visited both Ostuni and Matera from Alberobello. Alberobello to Matera is just 1+ hour drive. Alberobello to Ostuni is maybe 50 min drive. And next to Alberobello is also Locorotondo (very quaint) less than 10 min drive away.

Thanks for the info Roberto!

Yeah I can even find direct bus from Osturi to Alberobello.

But Alberobello to Matera, always have to transfer in Bari with public transportation.

I do not need a car in Alberobello or Matera correct? Both town are walkable, it is just the commute in between. Thinking about renting the car for one day.

rome2rio.com is always my first go to site.

I spent a month in the boot or Puglia or Southern Italy all with public transpo even in November when it was supposedly a tad more difficult, so definitely can be done.

Thanks Janet!
Were you in Matera or Alberobello by chance?

I would rent a car at the airport (Bari or Brindisi) and keep it for the whole trip, since you don't seem to have a problem renting a car for a day. You won't need it for Matera or any town, but it will make your transfers much more efficient and you'll be able to add more places to visit. If you base yourself in Alberobello or Locorotondo, you can visit everything from one location without needing to change hotels. All the localities below can be visited in a day trip, unless you want to spend the night a some specific locations. Alberobello is very central to many.
Alberobello to Matera = 70 min drive
Alberobello to Locorotondo = 5 min drive
Alberobello to Ostuni=45 min drive
Alberobello to Polignano=40 min drive
Alberobello to Monopoli=30 min drive
Alberobello to Martina Franca= 20 min drive

Depending on the length of your vacation, you might even be able to add more destinations, like Lecce, Otranto, etc.
If you stay in Bari (at the end or the beginning of your trip) make sure to rent after or before staying in Bari, since a car in the city center of Bari is a hassle.

That is really good to know. thanks Roberto!

My husband and I did three weeks in Puglia, all by public transportation. Was it slower than having a car? Yes, but we had the time and a disinclination to drive. We stayed in Matera and Alberobello and visited Bari and Brindisi on day trips. We didn't make it to Ostuni, which was the one place it would have eased things to be driving.

This is good to know--March 2027 we'll take the train from Rome after a couple weeks there, and explore Puglia. I imagine we'll base in Bari and take daytrips by train to Lecce & Polignano a Mare. And rather than try to do it on our own, I may hire a guide out of Bari to help us explore Matera & Alberobello. Fly out of Bari.

Hi Janet! Thanks for sharing!
Were you transfer in Bari when going to Matera and Alberobello ?

Sounds nice Jay! I will be happy to share more once I am back.
2027!! you are planning ahead!

This is janet_kupfer, not Janet from Boise or lower case janet---

Matera was lovely, very walkable, had a great apt, large, with an additional bathroom upstairs where the bedroom was!

I went to Alberobello from Polignano al Mare. Lesson learned: I scoped out the what I thought was the bus stop to go to Locotorondo first then Alberobello. I was on the wrong side of the street, the bus was so off schedule I did not know if it was earlier than the scheduled time or late. As he was zipping on down the street there was no one waiting for his bus at the bus stop, I missed the extremely tight connection to get to Loco and then onto Alberobello. Alberobello was my destination but I learned now to always know which direction I need to go in not the direction I think the bus should be going in. Not my first rodeo but I seem to learn something new every trip.

Guess we all learn something new during the trip and that is what make it intriguing :)
That's nice to know. Thanks Janet!