In developing countries, cowpea or Vigna unguiculata L. (Walp) is one of the staple foods of the population with cowpea seeds that are rich in proteins, carbohydrates and vitamins. Indeed, cowpea is attacked by a wide spectrum of insect pests, the Bruchidae Coleoptera, including Callosobruchus maculatus Fabricius. The conséquence of these attacks is the qualitative and quantitative decrease of the harvested or stored seeds due to the emergence of the flight morph from the flightless morph. The present work aims to determine the genetic diversity between the two morphotypes of Callosobruchus maculatus in Senegalese populations in order to develop more efficient control methods against this insect pest of cowpea seeds in storage. To carry out this work, we sampled twenty-nine (29) individuals constituting the flight morph population and nineteen (19) individuals forming the flightless morph population. After extraction, purification and DNA recovery using the Standard Qiagen method (Qiagen Kit), the Cytochrome B gene was amplified to obtain the studied sequences. However, flightless morphotypes are more monomorphic than the flight morph populations which are then more polymorphic because of a high rate of mutation per transition. On the other hand, the high rate of mutation per transversion in the flightless morph population could be favorable to a change of aminoacids and corresponding protein such as enzymes involved in metamorphosis. Indeed, this genetic diversity associated with haplotypic diversity could be a source of weak to moderate differentiation and this result was confirmed by the inter-population genetic distance which is greater than the intra-population genetic distance. This result would be partly due to environmental pressures leading to a weak genetic structuring between the two morphotypes according to the AMOVA values which present a significantly high percentage of variance. The neutrality tests and the Bayesian tree confirm this differentiation between the two morphotypes linked to an important gene flow within the total population. This suggests that some heterozygous flightless morph individuals have genetic predispositions to become flight morph individuals with the evolution of environmental conditions combined with the demographic and the maternal effect related to the female’s choice of spawning.