Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disease, caused by germline mutations in DNA mismatch-repair genes (MMR). These mutations lead to microsatellite instability (MSI). It has been found that the MSI is not confined to the setting of hereditary disease and may be seen in approximately 12-17% of the sporadic CRCs. In 1998 a National Registry for CRC was instituted in Queen Giovanna Hospital, Sofia. A total of 150 patients have been selected for MSI analysis and 25 tumors showed to be unstable, 14 with loss of heterozygosity (LOH). These tumors were further analyzed for MLH1 promoter hypermethylation and a significant association between this epigenetic change and MSI/LOH sporadic cases. We proposed this method as a step that follows the analysis for MSI and prior to the screening for MMR mutations. The mutation screening detected four known and two novel mutations, one unpublished and four known intronic polymorphisms in both hMLH1 and hMSH2 genes. The use of IHC analysis has been found effective in the investigation of some unclear molecular variations.We developed an efficient diagnostic strategy for HNPCC testing and the mutation status of 80% MSI HNPCC cases could be detected.