We have examined allelic variation at 23 loci coding for enzymes and nonenzymatic proteins in 14 species of the rodent genus Peromyscus. Five levels of differentiation in the process of speciation are recognized among these taxa. Degree of genomic modification was analyzed separately for 'fast' evolving loci and 'slow' evolving loci using Nei's (1972) measure of genetic distance, D. Little genic differentiation has occurred between local populations of P. boylii and P. pectoralis (D̄ = 0.030), with most of the divergence occurring among the 'fast' evolving loci. Average genetic distance between subspecies of P. boylii and P. pectoralis is 0.052, with limited genic divergence among 'slow' evolving loci in P. pectoralis. Sibling species and semispecies show a greater degree of differentiation, with D̄'s for each of 0.057 and 0.178, respectively. Allelic substitutions have not accumulated appreciably more among sibling species than among subspecies of Peromyscus, while differentiation among semispecies is approximately triple that of subspecies. This is best explained by different modes of speciation at these two levels. Well-differentiated species of the boylii species group have accumulated about 33 allelic substitutions per 100 loci since divergence, with nearly equal values of D̄ for both 'fast' and 'slow' evolving loci. During the initial stages of divergence, most of the reduction in genic identity is produced by allelic substitutions at 'fast' evolving loci. Once two taxa no longer share any alleles at a locus, additional allelic substitutions at that locus contribute nothing to overall level of divergence. During later stages of speciation, i.e., semispecies and well-differentiated species, divergence increases among 'slow' evolving loci. Values of D for various levels of differentiation in Peromyscus are not consistent with those of other taxa at comparable levels, about one third as great in some cases. Biochemically, Peromyscus, and rodents in general, appear to be conservative, with speciation occurring with major shifts to different adaptive zones with concomitant morphological and often chromosomal changes.