We compared and contrasted the physical structure of male "courtship" calls of 59 subterranean mole rats belonging to the Spalax ehrenbergi superspecies in Israel, comprising 11 populations of four chromosomal species (2N = 52, 54, 58, and 60). We also conducted behavioral auditory discrimination tests of 144 females of the four species in the laboratory. The results indicate that each chromosomal species has a vocal dialect significantly different from all others, although the call of 2N = 60, the last derivative of speciation, is not yet fully differentiated. Females of 2N = 52, 54, and 58 preferred their homospecific mates' calls, whereas females of 2N = 60 did not. We conclude that call differentiation builds up gradually and provides an efficient ethological reproductive premating isolation mechanism between the emerging species in the active speciation of mole rats in Israel.