Chromosomes from 14 species representing the six subgenera of the ground squirrel genus Spermophilus are evaluated as indicators of taxonomic relationships. The diploid number and karyotype of S. franklini (2n = 42), subgenus Poliocitellus, are described here for the first time, together with supplementary chromosomal characters from S. beecheyi (2n = 38) and S. variegatus (2n = 38), subgenus Otospermophilus, and S. lateralis (2n = 42), subgenus Callospermophilus. Chromosomal characters are diagnostic at the subgeneric level in Poliocitellus, Otospermorphilus, Xerospermophilus, Callospermophilus, and Ictidomys. The subgenus Spermophilus cannot be differentiated at this level because of a striking degree of interspecific variation (2n = 30–46). An evaluation of possible mechanisms for karyotype evolution within the genus Spermophilus provides additional taxonomic data. The subgenera Ictidomys and Poliocitellus appear to share an equally close chromosomal relationship to Spermophilus. Because more complex chromosomal rearrangements must be postulated to explain the differences observed between Callospermophilus, Otospermophilus, and Xerospermophilus greater evolutionary divergence may exist between these taxa than between Ictidomys, Spermophilus, and Poliocitellus.