Evidence is presented for six opossum ALDH1A genes, including four ALDH1A1-like genes on chromosome 6 and ALDH1A2- and ALDH1A3-like genes on chromosome 1. Predicted structures for the opossum aldehyde dehydrogenase (ALDH) subunits and the intron-exon boundaries for opossum ALDH genes showed a high degree of similarity with other mammalian ALDHs. Phylogenetic analyses supported the proposed designation of these opossum class 1 ALDHs as ALDH1Alike, ALDH1A2-like, and ALDH1A3-like and are therefore likely to play important roles in retinal and peroxidic aldehyde metabolism. Alignments of predicted opossum ALDH1A amino acid sequences with sheep ALDH1A1 and rat ALDH1A2 sequences demonstrated conservation of key residues previously shown to participate in catalysis and coenzyme binding. Amino acid substitution rates observed for family 1A ALDHs during vertebrate evolution indicated that ALDH1A2-like genes are evolving slower than ALDH1A1- and ALDH1A3-like genes. It is proposed that the common ancestor for ALDH1A genes predates the appearance of birds during vertebrate evolution.