Differentiation of traits among populations can evolve by drift when gene flow is low relative to drift or selection when there are different local optima in each population (heterogeneous selection), whereas homogeneous selection tends to prevent evolution of such a differentiation. Analyses of geographical variations in venom composition have been done in several taxa such as wasps, spiders, scorpions, cone snails and snakes, but surprisingly never in parasitoid wasps, although their venom should constrain their ability to succeed on locally available hosts. Such a study is now facilitated by the development of an accurate method (quantitative digital analysis) that allows analyzing the quantitative variation of large sets of proteins from several individuals. This method was used here to analyse the venom-based differentiation of four samples of Leptopilina boulardi and five samples of L. heterotoma from populations along a 300 km long south-north gradient in the Rhône-Saône valley (South-East of France). A major result is that the composition of the venom allows to differentiate the populations studied even when separated by few kilometers. We further analyzed these differentiations on the populations (reared under similar conditions to exclude environmental variance) with a QST analysis which compared the variance of a quantitative trait (Q) among the subpopulations (S) to the total variance (T). We also used random forest clustering analyses to detect the venom components the most likely to be adapted locally. The signature of the natural selection was strong for L. heterotoma and L. boulardi. For the latter, the comparison with the differentiation observed at some neutral markers revealed that differentiation was partly due to some local adaptation. The combination of methods used here appears to be a powerful framework for population proteomics and for the study of eco-evolutionary feedbacks between proteomic level and population and ecosystem levels. This is of interest not only for studying field evolution at an intermediate level between the genome and phenotypes, or for understanding the role of evolution in chemical ecology, but also for more applied issues in biological control.