I want to configure a 2 user multiseat in Linux.
Is it possible to configure keyboard and mouse for second seat with a USB hub on a single USB port or I need to connect each of them to a separate USB port?
The hub is irrelevant. You'd normally identify the device corresponding to each seat by its serial number. While you could identify the device by which hub it's connected to, and it might be convenient in some setups, it's more complicated, you don't have to do it this way.