The two genetically unlinked histone H2B genes isolated from Saccharomyces cerevisiae have been sequenced. The genes encode H2B proteins that are 130 amino acids in length and that differ by 4 amino acids. The changes betwen them are Ala leads to Ser, Lys leads to Ala, Thr leads to Val and Ala leads to Val at amino acid positions 2, 3, 27 and 35, respectively. A comparison of yeast H2B histones with those of higher eucaryotes demonstrates a high degree of homology clustered mainly at the carboxyl terminus. There is extensive base substitution between the two H2B genes in nucleotides that do not affect the amino acid sequence. DNA prelude sequences show 64% divergence. The coding regions differ at 49 of the 390 bases (12.6% divergence). 41 of these changes are in silent positions. By using the number of amino acid differences in the proteins we estimate that the two H2B genes are the result of an ancient duplication event.