Two DNA-based assays were developed for identification of the H2 alleles present in the 12 standard mouse MHC haplotypes H2b, H2d, H2f, H2j, H2k, H2p, H2q, H2r, H2s, H2u, H2v and H2z. The assays utilized polymerase chain reaction (PCR) amplification of a short stretch of genomic DNA including a highly polymorphic microsatellite from the second intron of the class II Eb gene within the murine major histocompatibility complex. The H2 Eb alleles were discerned by restriction fragment length polymorphism (RFLP) and heteroduplex analyses. For RFLP analysis amplified DNAs were digested with the restriction endonuclease Fnu4HI which delineated seven of the 12 alleles. A distinct pattern was obtained for the haplotypes H2d, H2j, H2k and H2p, whereas a group specific but distinct pattern was obtained for each of the three groups H2b, H2r and H2v, H2f, H2q and H2s, H2u and H2z. Heteroduplex analysis using a pair of haplotypes at a time helped further discriminate H2q from H2f or H2s. More importantly, heteroduplexing was quite informative in delineating the identity or disparity between two given haplotypes in a single step of PCR amplification. Both the RFLP and heteroduplex analyses are extremely sensitive and simple to operate, and since the target is genomic DNA, they can be carried out using any cell or tissue type.