Two groups of Anopheles claviger sensu stricto Meigen (Diptera: Culicidae) were recently found in France, representing unclear genetic entities. To better understand this situation, sampling was extended to 13 European countries, and 47 samples were analyzed by investigating the polymorphism of 11 autosomal and 1 sex-linked allozyme loci. Genetic differentiation, as measured by F(st), between these two groups was confirmed, with no isolation by distance within each group. Among the twelve loci studied, none had diagnostic alleles. Group I is mainly located in western Europe (UK and south-west of France), and Group II covers eastern France and eastern and northern Europe. Intermediate populations, sampled at the overlapping range between them, do not display a heterozygote deficit, suggesting that these two groups are probably not genetically isolated. The origin of each group and its biological significance is discussed within the context of differentiation in refugia during recent glaciations.