Anguina agrostis, Anguina tritici and Anguina wevelli are important pests of agriculture. However, only juveniles are generally found in seed galls and similar in morphology. Three pairs of primers, named as AgrF₁/AgrR1, TriF₁/TriR1 and WevF₁/WevR1, were designed based on rDNA-ITS nucleotide sequences of A. agrostis, A. tritici and A. wevelli. PCR techniques were developed and improved for detection of the three species with DNA extrated from single juvenile as templates. The amplified products for A. agrostis, A. tritici and A. wevelli were 499, 617 and 377 bp in size and showed a strong unique band in electrophoresis respectively. This study indicated that the multiple-PCR technology could satisfy the need of detection of A. agrostis, A. tritici and A. wevelli.