Mus terricolor I, II and III are the three chromosomal species which differ in stable autosomal short-arm heterochromatin variations established in homozygous condition. Analysis of meiosis in the laboratory-generated F1 male hybrids from crosses (both ways) between M. terricolor I and II and between M. terricolor I and III shows high frequencies of pairing abnormalities at pachytene. The backcross (N3 generation) male hybrids between M. terricolor I and II have meiotic abnormalities as in the F1 male hybrids, though to a lesser extent. They show difference in pairing abnormalities in the different karyotypic forms; the backcross hybrids heterozygous for the heterochromatic short arms have more anomalies compared to the homokaryotypic hybrids. This suggests a negative influence of the heterochromatin heterozygosity in meiotic pairing. The results indicate a role for heterochromatin variations in the development of a reproductive barrier in the speciating M. terricolor complex.