Distal-less (Dll) protein is the first genetic signal for limb formation to occur in the developing zygote in insects. The function seems to be different across various organisms, like the Dll in butterflies are not only involved in limb formation but also take part in eyespot and wing pattern formation. Hence, the study of the sequence variation of the Dll protein of different insects might help us in better understanding of its evolutionary divergence and in turn its function in different insects. The sequence of Dll protein were retrieved from the NCBI database and was used to study its relationships among other insects species using MEGA 4.0 and analysis of the physicochemical properties was done using a computational tool called PROT-PROP. The Dll protein in the insects showed variations (31-94% identity) in their sequences when BLAST was performed, but the homeobox domain exhibiting helix-turn-helix (HTH) was found to be conserved. Presence of motifs with identical amino acid sequence and presence of regions with poly-amino acids might be the reason for the differences in the role of Dll in different insects. In the phylogenetic tree, insects belonging to the same order were found to cluster together and exhibit genetic relatedness.