Correlation between RAS gene mutation and microsatellite instability (MSI) status in cancer tissues and clinicopathological parameters of patients with stage III colorectal cancer (CRC) were investigated. Tissues were collected from 180 patients diagnosed with stage III CRC in the Department of Gastrointestinal Surgery of the Fourth Hospital of Hebei Medical University from 2012 to 2016. RAS gene mutations in paraffin sections were detected by PCR and Sanger sequencing. Expression of mismatch repair proteins MLH1, MSH2, MSH6 and PMS2 was detected by immunohistochemistry, and MSI status was determined based on the positive and negative expression combinations of the above proteins, and the correlation with clinicopathological parameters of CRC was analyzed. Mutation rates of KRAS and NRAS were 48.33% (87/180) and 2.78% (5/180), respectively. Mutation rate of p.G12D in codon 12 of exon 2 in KRAS gene was the highest (31/87, 35.63%). Mutation rate of p.G12D in codon 12 of exon 2 in NRAS gene was the highest (2/5, 40%). Mutation rate of KRAS gene in right colon was higher than that in left colon and rectum (p<0.05), and mutation rate in N2b phase was higher than that in N2a and N1 phases (p<0.01). In low degree of microsatellite instability (MSI-L) and high degree of microsatellite instability (MSI-H) status, negative MKH1 protein expression was dominant (18/32, 56.25%). MSI-H in CRC patients aged ≥50 years was higher than that of CRC patients <50 years. Rates of MSI-H in N1, N2a, and N2b were 1.75, 12.82, and 1.11% (p<0.05). Mutation rate of KRAS gene in MSI-H status of stage III CRC patients was significantly higher than that in MSI-L/microsatellite stability (MSS) (p<0.05). Mutation of RAS gene and the status of MSI are involved in the occurrence and development of stage III CRC. Detection of RAS gene has important significance for the individual treatment of CRC in clinic.