After the initial development of a human immunodeficiency virus type 1 (HIV-1) subtype-screening tool by nested multiplex PCR, we further improve it through the redesign of subtype-specific primers based on subtype signature pattern (SSP) analyses and optimization of the PCR conditions. Extracted RNA from plasma samples was used in reverse transcription and the cDNA products were added to the first round PCR, in which universal primers in the gag region were used to detect HIV-1 M group isolates. In the second round of PCR, three pairs of subtype-specific primers, detecting subtypes B, C, and CRF01-AE, were added in one tube. Subtype determination was based on the different size of PCR products on the agarose gel electrophoresis. An additional set of primers detecting only the prevalent recombinant strains CRF07-BC and CRF08-BC was used to discriminate CRF07- and CRF08-BC from pure subtype C. Testing for all kinds of HIV subtype reference strains indicated that this assay was applicable. A panel of 252 HIV-positive samples and 30 HIV-negative samples was further used to evaluate and validate this assay. Compared to the assay of sequence-based phylogenetic analysis, the newly developed assay has an adequate designated subtype sensitivity, 93.2% (69 of 74) for subtype B, 95.1% (117 of 123) for subtype C, 94.0% (47 of 50) for CRF01-AE, and 95.0% (115 of 121) for CRF07-BC and CRF08-BC. Most importantly, the intersubtype specificity of the assay was found to be 100%. The assay specificity was also found to be 100% when used to test 30 HIV-negative samples. The average reproducibility was 96.0% for subtype B, 96.7% for subtype C, and 95.0% for CRF01-AE. We have developed a simple, rapid, and low cost assay for screening subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC in China.