Alvinella pompejana lives on the top of chimneys at deep-sea hydrothermal vents of the East Pacific Rise. It is thought to be one of the most thermotolerant and eurythermal metazoans. Our experimental approach combines methods of population genetics and biochemistry, considering temperature as a potential selective factor. Phosphoglucomutase (Pgm-1 locus) is one of the most polymorphic loci of A. pompejana and exhibits four alleles, from which alleles 90 and 100 dominate with frequencies of approximately 0.5 in populations. Results from previous studies suggested that allele 90 might be more thermostable than allele 100. Significant genetic differentiation was found by comparing contrasted microhabitats, especially the young, still hot, versus older and colder chimneys, with allele 90 being at highest frequency on young chimneys. Moreover the frequency of allele 90 was positively correlated with mean temperature at the opening of Alvinella tubes. In parallel, thermostability and thermal optimum experiments demonstrated that allele 90 is more thermostable and more active at higher temperatures than allele 100. This dataset supports an additive model of diversifying selection in which allele 90 is favoured on young hot chimneys but counterbalanced over the whole metapopulation by the dynamics of the vent ecosystem.