Background: Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infections is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis ofStrongyloides stercoralis infection by detection of copro-DNA in stool samples.Methods: A total of 782 fresh stool samples were collected and examined by agar plate culture.Among those sixteen stool samples, which confirmed to be infected withS. stercoralis were examined as positive control to set up each single and nested PCR, using two primer sets designing to amplify partial ribosomal DNA ofS. stercoralis genome. Since, single PCR method yielded higher efficacy in detecting positive samples, in the second step, 30 stool samples, which found negative forS. stercoralis by agar plate culture of single stool sample, were examined by single PCR. Data analysis was performed using McNemar's χ2 test, with consideration of a P value of<0.05 as indication of significant difference.Results: In amplification of DNA extracted from stool samples, single PCR detected S. stercoralis DNA target in all 16 positive samples, while nested PCR amplified DNA in only 75% of samples. In the second step, single PCR amplifiedS. stercoralis extracted DNA in 5 out of 30 samples which were negative by coproculture.Conclusion: Single PCR method amplifying a short (100bp) target represented more efficacies for detection ofS. stercoralis in faecal examination compared to agar plate culture and nested PCR, which amplified longer target.