Drosophila has been shown to be the best model system for the studies to unravel and to understand various aspects of genetic variations and its role in evolution. The nature and extent of genetic differences in populations has been analysed using Drosophila at different levels namely morphological, cytogenetic, chromosomal, biochemical, behavioural and ecological studies. This book is related to the study of nature and extent of intra and interspecific differentiation at ecological and behavioural level by analyzing the pupation site preference (PSP) or pupation site choice of the Drosophila larvae. First part deals with the effect of abiotic factors such as water, fruits, substrates and temperature on larval pupation site preference in Drosophila belonging to melanogaster, ananassae, subgroup of melanogaster species group, virilis and repleta group. Last part deals with the analysis of pupation site preference in relation to different biotic factors like larval density, size and weight, pattern and path length of locomotion.