Abstract: PAU genes constitute the largest gene family in Saccharomyces cerevisiae with 24 members mostly located in the subtelomeric region of chromosomes. Little information is available about PAU genes, other than expression data for some members. In this study, we systematically compared the sequences of all 24 members, examined the expression of PAU3, PAU5, DAN2, PAU17 and PAU20 in response to stresses, and investigated the stability of all Pau proteins. The chromosomal localization, synteny and sequence analyses revealed that PAU genes could have been amplified by segmental and retroposition duplication through mechanisms of chromosomal end translocation and Ty-associated recombination. The coding sequences diverged through nucleotide substitution and in/del of 1 to 4 codons, thus causing changes in amino acids, truncation or extension of Pau proteins. Pairwise comparison of non-coding regions revealed little homology in flanking sequences. All 24 PAU promoters contain a TATA box, and 22 PAU promoters contain at least one copy of the anaerobic response element and the aerobic repression motif. Differential expression was observed between PAU3, PAU5, PAU17, PAU20 and DAN2 in response to stress with PAU5 having the highest capacity to be induced by anaerobic conditions, low temperature, and wine fermentations. Furthermore, Pau proteins with 124 aa were less stable than those with 120 or 122 aa. Our results indicate that duplicated PAU genes have been evolving, and the individual Pau proteins might possess specific roles for the adaptation of S. cerevisiae to certain environmental stresses.