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Activating mutations of K-ras have been described in approximately 40% of patients with colorectal cancer, and are associated with resistance to epidermal growth factor receptor-targeted antibodies, such as cetuximab and panitumumab. Cost-effective and easy methods to determine K-ras mutations are urgently needed. Samples from 31 patients were tested. In laboratory 1, a real-time polymerase chain reaction (PCR)-based technique was used. All samples (n=31) were additionally tested using a restriction fragment length polymorphism (RFLP) analysis in laboratory 2. All results were confirmed by direct sequencing. In the first run, a concordance of real-time PCR and RFLP was observed in 77.4% (24 of 31) of samples. After resampling and reevaluation, a concordance of 93.5% (30 of 31) could be achieved. One of 7 (6.5%) initial discordant cases showed a mutation using real-time PCR and no mutation using RFLP, but the mutation was confirmed by direct sequencing. Real-time PCR and RFLP can be considered as valid K-ras mutation detection techniques. However, in patient probes with lower amounts of tumor cells and wild-type K-ras, reanalysis of further tumor tissue is recommended.