Multigene families are observed in all genomes sequenced so far and are the reflection of key evolutionary mechanisms. The DUP240 family, identified in Saccharomyces cerevisiae strain S288C, is composed of 10 paralogs: seven are organized as two tandem repeats and three are solo ORFs. To investigate the evolution of the three solo paralogs, YAR023c, YCR007c and YHL044w, we performed a comparative analysis between 15 S.cerevisiae strains. These three ORFs are present in all strains and the conservation of synteny indicates that they are not frequently involved in chromosomal reshaping, in contrast to the DUP240 ORFs organized in tandem repeats. Our analysis of nucleotide and amino acid variations indicates that YAR023c and YHL044w fix mutations more easily than YCR007c, although they all belong to the same multigene family. This comparative analysis was also conducted with five arbitrarily chosen Ascomycetes-specific genes and five arbitrarily chosen common genes (genes that have a homolog in at least one non-Ascomycetes organism). Ascomycetes-specific genes appear to be diverging faster than common genes in the S.cerevisiae species, a situation that was previously described between different yeast species. Our results point to the strong contribution, during DNA sequence evolution, of allelic recombination besides nucleotide substitution.