OBJECTIVE: To evaluate the utility of an automated insulated isothermal PCR (iiPCR) system for rapid and reliable on-site detection of African swine fever virus (ASFV) in swine biological samples. SAMPLE: Lymph node, tissue homogenate, whole blood, serum, spleen, and tonsil samples collected from swine in North and South Vietnam. PROCEDURES: Analytic sensitivity of the iiPCR system was determined by serial dilution and analysis of 2 samples (swine tissue homogenate and blood) predetermined to be positive for ASFV. Analytic specificity was assessed by analysis of 2 samples predetermined to be negative for ASFV and positive or negative for other swine pathogens (classical swine fever virus, porcine reproductive and respiratory syndrome virus, foot-and-mouth disease virus, and porcine circovirus type 2). Diagnostic performance of the iiPCR system for detection of ASFV was determined by analysis of the various tissue sample types. For all tests, a real-time PCR assay was used as the reference method. RESULTS: The iiPCR system was able to detect ASFV in swine blood or tissue homogenate at dilutions up to 106, whereas the real-time PCR assay was able to detect dilutions of up to 105 or 106. The iiPCR system had high analytic specificity for detection of ASFV versus other swine pathogens. Between 97% and 100% agreement was found between results of the iiPCR system for the various tissue samples and results of real-time PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE: The evaluated iiPCR system was found to be a rapid, reliable, and sample-flexible method for ASFV detection and may be useful for disease surveillance and quarantine in national strategies for early ASF control.