Previous small scale sequencing studies have indicated that DNA Polymerase β variants are present on average in 30% of human tumors of varying tissue origin. Many of these variants have been shown to have aberrant enzyme function in vitro and to induce cellular transformation and/or genomic instability in vivo, suggesting that their presence is associated with tumorigenesis or its progression. In this study, the human POLB gene was sequenced in a collection of 134 human colorectal tumors and was found to contain coding region mutation(s) in 40% of the samples. The variants map to many different sites of the Pol β protein and are not clustered. Many variants are nonsynonymous amino acid substitutions predicted to affect enzyme function. A subset of these variants was found to have reduced enzyme activity in vitro and failed to fully rescue Pol β deficient cells from methyl methane sulfonate (MMS) induced cytotoxicity. Tumors harboring variants with reduced enzyme activity may have compromised base excision repair (BER) function, as evidenced by our MMS sensitivity studies. Such compromised BER may contribute to tumorigenesis by leading to an increase in mutagenesis or genomic instability. Our results also indicate that tumors expressing Pol β variants may exhibit altered responses to DNA damage based therapies.