Abstract: ABSTRACT: BACKGROUND: Genes continuously duplicate and the duplicated copies remain in the genome or get deleted. The DAL5 subfamily of transmembrane transporter genes has eight known members in S. cerevisiae. All are putative anion:cation symporters of vitamins (such as allantoate, nicotinate, panthotenate and biotin). The DAL5 subfamily is an old and important group since it is represented in both basidiomycetes (mushrooms) and ascomycetes (yeast). We studied the complex evolution of this group in species from the kingdom of fungi particularly the ascomycetes. RESULTS: We identified numerous gene duplications creating sets of orthologous and paralogous genes. In different lineages the DAL5 subfamily members expanded or contracted and in some lineages a specific member could not be found at all. We also observed a close relationship between the gene YIL166C and its homologs in the Saccharomyces sensu stricto species and two "wine spoiler" yeasts Dekkera bruxellensis and Candida guilliermondi, which could possibly be the result of horizontal gene transfer between these distantly related species. In the analyses we detect several well defined groups without S. cerevisiae representation suggesting new gene members in this subfamily with perhaps altered specialization or function. CONCLUSIONS: The transmembrane DAL5 subfamily was found to have a very complex evolution in yeast not seen before with intra- and interspecific duplications and unusual relationships indicating specialization, specific deletions and maybe even horizontal gene transfer. We believe that this group will be important in future investigations of evolution in fungi and especially the evolution of transmembrane proteins and their specialization.