Using bioinformatic methods we have detected the genes of 40 keratin-associated β-proteins (KAβPs) (β-keratins) from the first available draft genome sequence of a reptile, the lizard Anolis carolinensis (Broad Institute, Boston). All genes are clustered in a single but not yet identified chromosomal locus, and contain a single intron of variable length. 5′-RACE and RT-PCR analyses using RNA from different epidermal regions show tissue-specific expression of different transcripts. These results were confirmed from the analysis of the A. carolinensis EST libraries (Broad Institute). Most deduced proteins are 12–16 kDa with a pI of 7.5–8.5. Two genes encoding putative proteins of 40 and 45 kDa are also present. Despite variability in amino acid sequences, four main subfamilies can be described. The largest subfamily includes proteins high in glycine, a small subfamily contains proteins high in cysteine, a third large subfamily contains proteins high in cysteine and glycine, and the fourth, smallest subfamily comprises proteins low in cysteine and glycine. An inner region of high amino acid identity is the most constant characteristic of these proteins and maps to a region with two to three close β-folds in the proteins. This β-fold region is responsible for the formation of filaments of the corneous material in all types of scales in this species. Phylogenetic analysis shows that A. carolinensis KAβPs are more similar to those of other lepidosaurians (snake, lizard, and gecko lizard) than to those of archosaurians (chick and crocodile) and turtles. J. Exp. Zool. (Mol. Dev. Evol.) 314B:11–32, 2010. © 2009 Wiley-Liss, Inc.