Large amounts of genetic variation for preferred temperature was uncovered in natural populations of Drosophila immigrans and D. virilis. On average, the former preferred 17.8°C and the latter did 21.0°C. Most variation in D. immigrans was found among isofemale lines within geographic populations, with no significant difference among geographic populations. Both intra- and inter-populational differentiation were significant in D. virilis. No geographic cline in preferred temperature was observed in either species. The distribution of flies on the temperature gradient indicates that the temperature selection of D. immigrans is stronger than that of in D. virilis.