TCR diversity depends mainly on the hypervariable complementarity determining region 3 (CDR3) α and β loops generated by non-germline-encoded nucleotide insertions and/or deletions. It is known that the length of the CDR3β sequence shortens during the process of thymocyte development and that the extent of this shortening is strongly affected by germline-encoded Vβ segments or MHC haplotypes. To examine whether CDR3α shortens to the same extent as CDR3β and how it is affected by Vα segments or MHC haplotypes, we analyzed CDR3α length distributions in thymic CD4+CD8+ (DP), CD4+CD8− (CD4SP) and CD4−CD8+ (CD8SP) T cells of different strains of mice (C57BL/6, C.B10 and Balb/c). As expected, CDR3α shortening occurred in both the CD4SP and CD8SP cells of all strains tested and the extent of shortening varied considerably depending on Vα segment use. However, there was no correlation in the extent of CDR3α shortening in individual Vα segments between CD4SP and CD8SP cells. Interestingly, there was a significant correlation in the extent of CDR3α shortening among different strains of mice only in CD4SP but not CD8SP cells, independent of MHC haplotype. These results suggest that the extent of CDR3α shortening is primarily determined by germline-encoded Vα segments and affected by allelic variation of MHC class I but not of MHC class II. The present study showed that T cells with shorter CDR3α sequences are preferably selected for the functional TCR repertoire during thymocyte development, and this provides an intriguing insight into the interactions of the TCR and p-MHC ligand.