Environmental and social factors are among the most important drivers of evolution of the acoustic structure of calls in mammals. For nine vole species, we compared the values of acoustic variables (duration, fundamental frequency and peak frequency) and discretely varying categorical traits of ultrasonic calls (10–15 calls per pup, 905 in total) recorded from 2–5-day-old pups (7–10 pups per species, 87 in total) isolated from the nest for 2 min and then weighed and measured for body variables. The species were different in body size, in the acoustic traits of their calls, and in preferential call contour shapes, note compositions and nonlinear vocal phenomena. The acoustic structure of the ultrasonic calls was species-specific, enabling 67.8% discrimination of calls to correct species with discriminant analysis. Across species, call duration varied from 35 to 166 ms, the maximum fundamental frequency from 39.9 to 61.6 kHz and the minimum fundamental frequency from 23.1 to 43.1 kHz. For the five species, the pup ultrasonic calls were described for the first time. Only pup body size influenced the acoustic parameters of pup ultrasonic calls, correlating positively with call duration and negatively with frequency variables. In contrast, the phylogenetic relatedness, lifestyle (subterranean vs. surface-dwelling), species micro-habitat (tall-grass vs. sparse-grass) or family group composition were not responsible for the differences in the acoustic traits across the nine vole species. We provide a synthesis of hypotheses explaining acoustic variation across vole species.