SUMMARY Strongyloidiasis is frequently asymptomatic and diagnosis of latent infection is difficult due to limitations of current parasitological and serological methods. This study aimed to verify the use of conventional polymerase chain reaction (PCR) assay for molecular diagnosis of Strongyloides stercoralis infection. Fresh stool samples were obtained from 103 individuals: 33 S. stercoralis positive, 30 positive for other parasites and 40 negative for parasitological methods. These samples were examined by the Lutz, Rugai and agar plate culture methods and conventional PCR assay. Two sets of primers (S. stercoralis species-specific and genus-specific sets), located in the 18S ribosomal RNA gene, were used for PCR. Of the 33 samples positive for S. stercoralis by parasitological methods, 28 (84·8%) were also detected by PCR assay using species-specific primers and 26 (78·8%) using genus-specific primers. Among the stool samples negative by parasitological methods, seven (17·5%) were positive by PCR using species-specific primers and two (5·0%) using genus-specific primers. In conclusion, the conventional PCR assay described in this study using a species-specific primer pair provided a molecular method for S. stercoralis diagnosis in human stool samples.