As part of a study of the population biology of pine voles (Microtus (Pitymys) pinetorum), 65 voles were karyotyped from seven apple orchard populations in the eastern United States. All animals had 62 chromosomes. Voles from New Hampshire and New York had 62 autosomal arms, whereas those from Virginia and North Carolina had 64. Voles from one population in southern Pennsylvania had 62, 63, or 64 arms in approximate Hardy–Weinberg proportions. The polymorphism resulted from the presence or absence of secondary arms on the largest pair of autosomes. There appears to be no reproductive isolation between the cytotypes in the Pennsylvania population.Key words: Microtus pinetorum, Pitymys, chromosomal variation, karyotype.