Do my customers have to create an account on my portal before they are allowed to book?

No, they usually do not have to. Most portals allow a guest booking, where the customer enters the traveller details and pays without setting up a login, and the confirmation is sent to the email address they typed at the time. You can normally decide which way your own site works, so if you would rather every booking sat under a registered account, that can be switched on instead.

There is a trade off either way. Guest booking removes a step and tends to lose fewer customers part way through paying, which matters most on a site where people are booking once and not coming back. An account gives the customer somewhere to see their trips, download a voucher again if they lost the email, and save traveller details for next time, and it gives your staff a cleaner history of who booked what. Many portals keep both, with the guest given the option to turn the booking into an account afterwards.

What you cannot skip is the traveller information the supplier requires, and that is the same either way. Names as they appear on the passport, dates of birth where the fare needs them, and a contact email and phone number all still have to be collected, because they travel with the booking to the airline or the hotel. An account only decides whether those details are stored for reuse.

The next thing to do is look at the booking settings on your portal, decide whether guest booking suits the customers you actually get, and place one test booking each way so you can see what the customer receives in both cases.