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Described here an amplification-based Sequence Specific Oligonucleotide Probe Hybridization (SSOPH) assay for high resolution allotyping of four HLA DRB1*01 group alleles. A region within exon 2 of HLA DRB1 gene was amplified by using group specific biotin-labeled primers. The amplicons were then hybridized to immobilized oligonucleotide probes that were specific for HLA DRB1*01 group alleles (HLA DRB1*0101, *0102, *0103 and *0104). The Hybridized amplicons were detected by an enzymatic-colorimetric reaction. One hundred and fifty DNA samples were tested by this method, in parallel with PCR-SSP and DNA sequencing for demonstrating the accuracy of the Reverse Dot Blot (RDB). Results suggested that PCR-RDB is a rapid, accurate and cost effective method for high resolution HLA molecular typing in comparison to PCR-SSP and DNA sequencing.