Diagnosis and epidemiological analysis of human parvovirus B19 (hB19V) infections are essential for disease management in severely ill patients. This study aimed to evaluate the performance of an optimized NS1-VP1u nested PCR for detection and sequencing of viruses in clinical samples using 224 clinical and five reference samples. PCR sensitivity, specificity, and positive and negative predictive values were perfect (100%). While phylogenetic analysis of a 615 bp-long fragment demonstrated that the viruses in all of the samples belonged to genotype 1, this study confirmed that this optimized PCR could detect all known hB19V with high performance.