A new study design based on cross-validation of the age at the onset of rectal cancer and the differences between the frequency distributions of relevant genes in 2 groups was developed for identification of disease-related HLA. Patients with rectal cancer were recruited and their age at the time of the first surgery was recorded. The genetic variants of HLA-DQB1 were genotyped using an HLA-DQB1 PCR-SSP typing kit. Allele frequencies were compared with control population. The mean age of patients with and without the alleles was compared. The frequency values of HLA-DQB1*02 were 12.3% higher in the cancer group than in the control population (P < 0.05). The median ages of the subjects with and without HLA-DQB1*02 were 54.0 and 61.0 years, respectively, with significant difference observed between the ages for these groups (P < 0.05). The median ages of the subjects with and without HLA-DQB1*03 were 62.0 and 58.0 years, respectively, and a significant difference was observed. The cross-validation of the 2 above mentioned analytical results showed that a statistically significant difference was noted for HLA-DQB1*02 (P < 0.05), whereas no such statistically significant difference was observed for HLA-DQB1*03. HLA-DQB1*02 allele was related to cancer susceptibility. The new analysis method may be an efficient and reliable approach for the identification of disease-related HLA.