In this sentence, yes. Ποιος είναι; translates as "who is it?" (in the case someone knocks your door for example and you do not know the gender). So who is he? is ποιος είναι αυτός; to specify the gender. ποιος είναι αυτός is also "who is that/this" for males.
Yes, Greek has three genders: masculine (e.g. ο άντρας - the man, ο αέρας - the air), feminine (e.g. η γυναίκα - the woman, η πόλη - the city), and neuter (e.g. το παιδί - the child, το πάτωμα - the floor).