Larval pupation site preference (PSP) of different species of Drosophila was analyzed on fruit in the laboratory. The larvae of D. melanogaster, D. ananassae, D. virilis, D. novamexicana and D. hydei pupated on the surface of glass vials; D. simulans, D. yakuba, D. mauritiana and D. malerkotliana pupated in/on fruit; and D. rajasekari pupated on cotton plugs in all experiments. D. bipectinata larvae changed their preference from fruit in the control to glass surface for all of the fruits tested. The statistical analysis of PSP (glass and fruit) found a significant result in that compared to other species, D. mauritiana and D. ananassae preferred to pupate on cotton compared to the control.