Hereditary colorectal cancer (CRC) syndromes remain incompletely understood, with many cases lacking defined genetic causes. This study aimed to identify pathogenic mutations associated with hereditary CRC and explore their potential for targeted therapies.This observational study was conducted in Yunnan Province, China. We analyzed 43 individuals from 12 hereditary CRC families using whole-exome sequencing, screened 84 polyposis families and 310 sporadic CRC cases, and an expanded cohort of 285 individuals with potential hereditary CRC. A series of in vivo and in vitro assays were conducted to evaluate the mutation's effects on tumorigenesis.A germline heterozygous stop-gain mutation, p.Arg1953X in the POLQ (polymerase theta) gene, was identified in two CRC families, showing co-segregation with disease status. A third POLQ mutation-positive family was identified in the expanded validation cohort. Cells carrying the mutation showed potential of tumorigenesis. The mutation hyperactivates error-prone theta-mediated end-joining (TMEJ), leading to high tumor mutational burden (TMB) and resistance to DNA-damaging treatments. Indeed, the probands exhibited mismatch repair deficient/microsatellite instability-high status (dMMR/MSI-H) that indicates high TMB. Treatment with the POLQ inhibitor Novobiocin suppressed TMEJ activity and restored tumor sensitivity to DNA damage, providing a combined medication scheme for drug-resistant POLQ-type CRC.This study identifies POLQ as a pathogenic gene in hereditary CRC, unveiling a novel POLQ-type CRC driven by TMEJ hyperactivation. Screening for POLQ mutations in patients with hereditary adenomas or early-onset CRC would benefit early diagnosis and personalized therapy for POLQ-associated CRC, while further clinical validation is warranted.