Scabies is still a health problem in the world. In Indonesia, the prevalence of scabies in 2017 was 6% of the total population. The diagnosis of scabies can be established clinically by finding its four cardinal signs. However, mostly in practice, the clinical symptoms are atypical, resemble other manifestations of skin diseases, such as eczema and/or impetigo, or the patient presents with secondary infection and lichenification. This may cause misdiagnosis that leads to unfair treatment. Consequently, the patient does not recover and continues to be a source of infection for their environment. Since clinical and microscopic diagnosis is difficult, this study aims to evaluate molecular detection to identify scabies infestation in suspected patients and compare it to clinical diagnosis. The skin scrapings used as samples in this study were collected from Darul Ishlah Islamic Boarding School students. The subjects were selected using a cross-sectional survey formula to determine the sensitivity and specificity of the clinical diagnosis method compared to PCR, which was the targeted Cox-1 gene. Out of the 53 patients diagnosed clinically with scabies, 50 were S. scabiei positive on PCR examination, and 8 additional positive PCR results were from 35 clinically negative patients. When compared with the IACS 2020 criteria, a significant difference in proportion was discovered between clinical and molecular diagnoses. Therefore, the PCR sensitivity in diagnosing scabies infestation was 94.1%, 77.3% specificity, 87.5% accuracy, 86.2% positive predictive value, and 90% negative predictive value. The results showed that PCR can be used to detect scabies infestation as an evidence-based diagnostic tool.