Candida albicans strain SC5314 contains two ALS3 alleles, which differ in sequence with respect to the number of copies of the 108 bp tandem repeat sequence within the central domain of the coding region. One allele (ALS3(12)) has 12 tandem repeat copies while the other (ALS3(9)) has 9 copies. Wild-type C. albicans (ALS3(12)/ALS3(9)) and those containing various ALS3 alleles (ALS3(12)/als3Delta(9), als3Delta(12)/ALS3(9) and als3Delta(12)/als3Delta(9)) were assayed for adhesion to monolayers of cultured vascular endothelial and pharyngeal epithelial cells. These assays showed obvious adhesive function for the larger Als3p protein, compared to a minor contribution to adhesion from the smaller protein. These functional differences in strain SC5314 prompted examination of ALS3 allelic diversity across the five major genetic clades of C. albicans. This analysis focused on the number of copies of the tandem repeat sequence within the central domain of the coding region and showed a range of alleles encoding from 6 to 19 tandem repeat copies. Clades differed with respect to prevalent ALS3 alleles and allele distribution, but were similar for the mean number of tandem repeat copies per ALS3 allele. Analysis of allelic pairing showed clade differences and the tendency for C. albicans strains to encode one longer and one shorter ALS3 allele. The allelic variability observed for ALS3 and its functional consequences observed in strain SC5314 highlight the importance of understanding ALS allelic diversity in order to draw accurate conclusions about Als protein function.