Four highly conserved members of the skeletal protein 4.1 gene family encode a diverse array of protein isoforms via tissue-specific transcription and developmentally regulated alternative pre-mRNA splicing. In addition to the prototypical red blood cell 4.1R (human gene symbol EPB41,) these include two homologues that are strongly expressed in the brain (4.1N, EPB41L1; and 4.1B, EPB41L3) and another that is widely expressed in many tissues (4.1G, EPB41L2). As part of a study on the structure and evolution of the 4.1 genes in human and mouse, we have now completed the chromosomal mapping of their respective loci by reporting the localization of mouse 4.1N, 4.1G, and 4.1B, as well as human 4.1B. For the mouse 4.1 genes, Southern blot analysis of RFLPs in The Jackson Laboratory BSS interspecific backcross yielded the following assignments: 4.1N (Epb4.1l1,) chromosome 2; 4.1G (Epb4.1l2,) chromosome 10; and 4.1B (Epb4.1l3,) mouse chromosome 17. Human 4.1B was physically mapped to chromosome 18p11 using fluorescence in situ hybridization. All of the mouse genes mapped within or adjacent to regions of conserved synteny with corresponding human chromosomes. We conclude that a set of four paralogous 4.1 genes has been evolutionarily conserved in rodents and primates.