The systematic relationship of two species of the Peromyscus truei group (P. truei and P. difficilis) was analysed through the application of starch gel electrophoresis, numerical taxonomy, and chromosomal techiques. Of 20 loci examined, 11 were monomorphic in all populations, two exhibited variation in only two populations, and seven loci were polymorphic in two or more populations. The mean number of polymorphic loci per population was 0.186, the mean number of polymorphic loci per individual was 0.024, and the proportion of loci heterozygous per individual was 2.4%. Chromosomal forms of P. truei, P. t. gentilis (FN 54) and P. t. truei (FN 62), and P. difficilis, P. d. petricola (FN 56) and P. d. nasutus (FN 58), were consistent for their karyotypes throughout their geographic ranges. No chromosomal hybrids were detected. Numerical analysis of morphological characters and similarity values based on allelic frequencies utilizing Roger's coefficient (S) demonstrated a distinct seperation of karyotypic forms of P. truei (S = 0.902) and P. difficilis (S = 0.924) and were below the mean value of S for conspecifics (S = 0.950). All data indicates that each chromosomal form of the P. truei group examined represents four distinct species. The oldest available name for chromosomal forms of P. true with a fundamental number of 62 is Peromyscus truei Shufeldt (1885) and the oldest available name for chromosomal forms with a fundamental number of 54 is Peromyscus gratus Merriam (1898). The oldest available name for chromosomal forms of P. difficils with a fundamental number of 58 is PeromYSCus nasutus Allen (1891) and the oldest available name for chromosomal forms with a fundamental number of 56 is Peromyscus difficilis Allen (1891).