Antigenic variation, which is the result of amino acid substitution and genetic evolution, poses a major challenge in the development of vaccines against pathogens with unstable genomes, such as HIV-1 and Influenza. Thus, it is highly important to characterize the relationship of genetic evolution, antigenic variation and positional mutation (GAP) from the perspective of vaccine development. For this purpose, we introduce an automatic and simple GAP analysis approach, which is based on the fundamental concept of “mutational behavior” in genomic research, to predict the specificity- determining positions in protein families. This approach identifies cross-neutralization determinants by mutational behavior correlation to antigenic and genetic changes. Correlated mutation analysis and structure mapping further refine the cross-neutralization determinants. The usability of this approach is confirmed by analysis of an Influenza H3N2 crossreactive dataset.