The island of Madeira harbours highly differentiated Robertsonian (Rb) races of the house mouse (Mus musculus domesticus), carrying several unique Rb fusions. The reproductive and geographical isolation between these races is expected to restrict gene flow severely, and contribute to their genetic differentiation. In the present study, the chromosomal distribution and frequency of transcriptionally active nucleolar organizer regions (AgNORs) between five Madeiran races was studied. The results showed that AgNORs were present in the centromeric regions of five chromosome pairs. Differences in NOR distribution were apparent within and between races. Comparison between races showed that AgNOR variation occurred independently of the Rb or non-Rb state of the chromosomes and of the NOR-bearing status of the partner chromosomes. The results indicated that Rb fusion formation does not interfere with the activity of rDNA genes. In addition our data provided no evidence that the presence of AgNOR sites inhibits Rb fusion. A cluster analysis showed that AgNOR differentiation was only partly congruent with the chromosomal divergence and geographical proximity between races. In particular, parapatrically distributed races showing the lowest chromosomal divergence exhibited high levels of AgNOR differentiation. If confirmed, this suggests that premating isolation may be reinforcing a barrier to gene flow between the races. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 585–591.