We developed an adaptor ligation PCR-based microplate hybridization assay (MHA) to analyze the repertoires of mouse T-cell receptor (TCR) α- and β-chain variable regions (TCRAV and TCRBV). RNA is transcribed to cDNA and an adaptor is ligated to the 5′ end of the cDNA, which is then used as a template for PCR with an adaptor-specific 3′ primer and a constant region-specific 5′ primer. After hybridization of PCR products with TCRAV- and TCRBV-specific probes on the microplate, quantitative ELISA was carried out.The entire TCRAV or TCRBV repertoires could be analyzed using a single 96-well plate in triplicate and completed in less than 4 h. The assay results demonstrated the high level of specificity and reproducibility of this method. Furthermore, MHA results correlated well with those of fluorescence-activated cell sorting. This method may provide important information about various T-cell-associated diseases including autoimmune disease. The influence of the MHC on mouse TCR repertoires was next studied using the newly developed mouse TCRAV and TCRBV repertoire assay. The analysis in six strains showed no significant correlation between MHC haplotypes and TCRAV and TCRBV repertoires. However, large differences among strains was observed in TCRBV, but not in TCRAV repertoires. There were also large differences within same strain in TCRBV, but not in TCRAV repertoires, indicating differences in individuals independent of genetic factors. These data suggest that TCRBV repertoires are more susceptible than TCRAV repertoires not only to genetic factors but also some environmental factors.