House dust mites live in house dusts and affect the health of humans. Among the many species, Dermatophagoides farinae, D. pteronyssinus, and Euroglyphus maynei have been found to be commonly associated with Ig-E-mediated allergic diseases. As a result, there is increasing effort to develop methods for the diagnosis and treatment of diseases caused by these species. The purpose of the current study was to explore the evolutionary relationships among house dust mites. After adult D. farinae were separated and isolated for total RNA extract, the cDNA coding for Der f 1 and Der f 2 were cloned and sequenced. Then amino acid sequences for group 1 and 2 allergens of two of the most common house dust mites, D. pteronyssinus, E. maynei, were obtained from databases. Interestingly, homological analysis of amino acid sequences showed that both Der p 1 and Der p 2 from D. pteronyssinus had more similarities to Eur m 1 and Eur m 2, respectively, than they had to Der f 1 and Der f 2 from D. farinae. In the phylogenetic trees, D. pteronyssinus clustered with E. maynei, but not with D. farinae, although D. pteronyssinus and D. farinae belong to the same genus according to morphological taxonomy. It was previously assumed that D. pteronyssinus was more similar to E. maynei than to D. farinae at evolutionary levels.