Abstract: Introduction Much is known about mutations in colorectal cancer (CRC), but little is known about evolutionary paths connecting carcinomas to their ancestral adenomas. To investigate genetic alterations associated with the adenoma-carcinoma transition, we collected polyps isolated during routine colonoscopy which contained the first visible stages of colorectal neoplasia, so-called carcinomas in-situ. These samples provided us with the earliest window of transition. Material and methods We sequenced adenoma and carcinoma DNA separately using a CRC driver gene panel and compared single nucleotide variants (SNVs) and copy number alterations (CNAs). Using multi-regional sampling and a Bayesian Dirichlet clustering process we tracked sub-clonal progression pathways to CRC. In addition, we explored regional patterns of P53 using immunohistochemistry. Samples which revealed little discordance were additionally sequenced at the whole exome level. Results and discussions Our results demonstrate that recurrent genetic alterations at the chromosomal level can precede APC initiating mutations. We find adenoma heterogeneity to be extensive, providing a ‘playground’ for the initiation of carcinomas. Indeed, heterogeneity in driver genes such as RAS and PIK3CA appears to be greater in adenoma compared to carcinoma. Additionally, our study shows P53 to be associated with, but not sufficient for, adenoma to carcinoma progression. Conclusion Our work at the transition border between an adenoma and a carcinoma in the colon provides a further layer of complexity to the Vogelstein step-wise progression model of CRC.