Single nucleotide polymorphisms (SNPs) may contribute to the malignant process and may show clinicopathological importance as prognostic markers. The multidrug resistance gene MDR1 encodes a membrane transporter which confers cytostatic drug resistance in tumors and protects normal tissues from xenobiotics. We analyzed the C3435T SNP in the MDR1 gene which is associated with altered cellular drug uptake in matched tumor and normal tissues of 45 patients suffering from colorectal carcinoma. We have developed a highly sensitive matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method to survey the C3435T polymorphism in PCR-amplified fragments of the MDR1 gene. Thirteen patients were homozygous for C/C (29%), 15 were heterozygous (33%) and 17 were homozygous for T/T (38%). None of the tumor samples showed an altered SNP compared to their matched normal tissue samples. As analyzed by the Kruskall-Wallis test, none of the clinicopathological parameters was significantly associated with homo- or heterozygosity. The combination of PCR, allele-specific primer extension reactions and MALDI-TOF-MS offers a promising alternative method for genotyping the MDR1 gene especially for heterozygous situations. The inherent advantages of MALDI-TOF-MS based genotyping include its high molecular resolution, high signal-to-noise-ratios and reproducibility, combined with an excellent sensitivity. As none of the tumor samples showed an altered state compared to their matched normal tissue samples, the genotypic frequency of this polymorphism seems not to be altered during colorectal tumorigenesis.