IMO she'd started to wonder about it around the time (film version) that she met Cassie. Alma Jr would have remembered meeting Jack that one time, and both girls would have noticed their father acting differently after Jack drove away. Even his posture and facial expression would have caught their attention.
Also, we don't know what happened at Alma and Monroe's house after Ennis storms out that Thanksgiving, but Alma was clearly upset enough to make remarks the girls would have wondered about. And if she told Monroe any details about the fight, would the girls not try to eavesdrop? What they know at that point is that their father joined Alma in the kitchen right after dinner and just a few minutes later he abruptly leaves and she's yelling and smashing something.
Besides, Alma Junior and Jenny are not only from a very different generation but they also probably have much more contact with the larger world than Ennis did growing up. By the early 1980s, homosexuality was no longer the taboo subject it had been when Ennis and Jack were the same age, so it would be surprising if they didn't at least speculate about it.