Objective: To investigate K-ras gene mutation status and its relations with the clinicopathological characteristics in patients with colorectal cancer. Methods: The K-ras gene mutation status in the tumor tissues from 200 colorectal cancer patients were detected by Real-time fluorescence quantitative PCR (RT-qPCR) and direct sequencing, respectively. The results obtained by the two methods were compared and their significance was analyzed in conjunction with the main clinicopathological variables. Results: Of the 200 colorectal cancer specimens, 63 mutation cases were detected by RT-qPCR, and the mutation detection rate was 31.5%; 50 mutation cases were detected from the 169 specimens that were successfully sequenced by direct sequencing technique, and the mutation detection rate was 29.6%. The GGT→GAT at codon 12 was the most prevalent mutation and accounted for 34.9% (22/63) of the total mutation cases, followed by GGC→GAC at codon 13 that accounted for 28.6% (18/63) of the total mutation cases, while GGT→GCT at codon 12 was the rarest mutation that was not found in any case of the entire group (0/63). The concordance rate between two methods was 98%. K-ras gene mutation was significantly associated with the differentiation of the tumor (P<0.05), but not related to the sex, age, tumor location, lymph node metastasis or TNM stage of patients (all P>0.05). Conclusion: RT-qPCR is a rapid, sensitive and accurate method for detection of the K-ras gene mutation, and therefore provides reliable information for clinical applications of targeted therapy.