To study the pupation site preference which is an important larval behavior, pupation height was scored in Drosophila malarkotliana, D.bipectinata and D. parabipectinata using fructose sugar in culture media. Strains of these species were collected from Unchahar, Bachhrawan, Raebareli & Salon localities in Raebareli Districts. The mean pupation height in different strains of Drosophila malarkotliana ranged from 3.56 to 3.67, in D. bipectinata ranged from 4.58 to 5.02 and D.parabipectinata., ranged from 1.96 to 2.03. The analysis of variance and t-test were performed to test intra and interspecies in pupation height. These tests revealed significant variation among different strains of the species. Significant variations among male & female for pupation height the same species were also found in all species Drosophila. These observations provide evidence for ecogenetics variations in pupation height of three species of Drosophila. Variations among different strains of the same species in pupation height are attributable to genetic heterogeneity among the strains.