The location of rDNA on chromosomes has been found to be highly variable in different groups of insect families of various orders. Yet, in other insect families the rDNA position is relatively constant. This contrast so far has received limited attention. We investigated the rDNA position on mitotic chromosomes in 18 species of Drosophila, 16 of which are from the tripunctata radiation, subgenus Drosophila, through fluorescent in situ hybridization (FISH). All species showed fluorescent signals only on the sex chromosomes. On the X chromosome a single fluorescent mark, but with variable locations, was found. On the Y, we observed variation both in location and in number of fluorescent marks (from 1 to 5). This constancy of chromosome location, in contrast to the great variability found in other groups, is consistent with the work carried out in other species of Drosophila. This suggests that the presence of a nucleolus organizer region (NOR) on each sex chromosome is probably an ancestral condition in the genus. Moreover, this difference in the variation of rDNA position among groups points out an interesting evolution question, which deserves further study.