Recent research has demonstrated biologic plausibility for iatrogenic tumor seeding via colonoscopy as a cause of metachronous colorectal cancers (CRC). This study evaluated the association between biopsy of non-tumor sites after CRC biopsy and risk of metachronous CRC in a large community-based health care organization. This was a retrospective case-control study of adults with an initial CRC diagnosed by colonoscopy between January 2006 and June 2018 who underwent curative resection. Cases developed a second primary (metachronous) CRC diagnosed 6 months to 4 years after the initial CRC, and were matched by age, sex, diagnosis of inflammatory bowel disease, race, and ethnicity with up to 5 controls without a second CRC diagnosis. The exposure was biopsy in the colonic segment of the metachronous CRC (or corresponding segment in controls) after tumor biopsy, ascertained with blinding to case status. Associations were evaluated using conditional logistic regression and adjusted for potential cofounders. Among 14,119 patients diagnosed with an initial CRC during colonoscopy, 107 received a second CRC diagnosis. After exclusions for recurrent or synchronous CRC, 45 cases and 212 controls were included. There was no significant association between biopsy of non-tumor sites after initial CRC biopsy and risk of metachronous CRC in the segment of the additional biopsy site (adjusted odds ratio, 2.29; 95% confidence interval, 0.77-6.81). Metachronous cancers are not significantly associated with biopsy of non-tumor sites after biopsy of the primary cancer. Although the sample size does not allow definite exclusion of any association, these findings do not support iatrogenic tumor seeding as a common risk factor for metachronous CRC.