Homeobox DNA polymorphisms (restriction fragment length polymorphisms, RFLPs) were first found in 13 individuals, and then in 121 subterranean mole rats of the Spalax ehrenbergi superspecies in Israel. Spalacidae are adapted morphologically to life underground apparently since Oligocene times, 30 million years ago. The superspecies S. ehrenbergi comprises four chromosomal species (2n = 52, 54, 58, and 60) displaying active speciation and dynamic adaptive radiation into four climatic regimes. A Hox-1.1 and -1.5 polymorphism was found with EcoRI, where three of four individuals of the species 2n = 60 were lacking the diagnostic locus-specific band present in all other nine individuals tested in the first stage. Next we found monomorphism in 121 animals in Hox-2.1 digested with five six-base recognition endonucleases (EcoRI, HindIII, BstEII, KpnI, BamHI, and one four-base enzyme, TaqI). However, we found in the same 121 animals from 13 populations of the four species in Hox-3.1 species-specific TaqI polymorphism in two populations of 2n = 54, and HindIII polymorphism in five populations, three of 2n = 54 and two of 2n = 58. The homeobox polymorphisms found display significant correlations with the burrow microclimates of mole rats. Furthermore, they are significantly correlated primarily with diverse morphological variables, and secondarily with protein (allozyme) and DNA polymorphisms, but very little with physiological and behavioral variables. We suggest that in subterranean mole rats, the variation in microclimatic factors, particularly in more unpredictable xeric regions, may maintain genetic polymorphisms by diversifying selection in the developmental control homeobox genes Hox-1.1, -1.5, and Hox-3.1, affecting regulatory morphogenetic processes in evolution. By contrast, the Hox-2.1 monomorphism suggests strong purifying selection for conservation across the superspecies over long periods of evolutionary times. © 1992 Wiley-Liss, Inc.