Karyotypes were prepared from 146 individuals, representing nine populations evenly spaced along a 2,000-km north-south transect in Paraguay and Argentina, to determine the nature, extent, and pattern of chromosomal variation in Holochilus brasiliensis chacarius and H. vulpinus. Two distinct patterns of chromosomal variation characterized these two species. In H. brasiliensis, the diploid number (2n) ranged from 48 to 56 and the nombre fondamental (NF) from 57 to 63. Four classes of chromosomal variation were found in populations of H. brasiliensis: whole-arm Robertsonian (Rb) translocations, including Rb changes with monobrachial homology, variation in the number and kind of supernumerary (B) chromosomes, centromeric rearrangements (putative pericentric inversions), and variation in the amount of euchromatin. The amount of structural variation was uniformly high in all populations of H. brasiliensis sampled, and all rearrangements appeared to be in Hardy-Weinberg proportions, corroborating the hypothesis that chromosomal rearrangements are not strongly underdominant in this species. In H. vulpinus, 2n ranged from 35 to 39 and NF from 57 to 61. Two classes of variation were found in this species: variation in the number, but not the kind, of supernumerary chromosomes and variation in the amount of euchromatin.