Treatment of locally advanced rectal cancer includes chemotherapy, radiation, and surgery but patient responses to neoadjuvant treatment are variable. We have shown that rectal tumors are comprised of multiple genetically distinct sub-clones. Unique sub-clones within tumors may harbor mutations which contribute to inter-patient variation in response to neoadjuvant chemoradiotherapy (nCRT). Analysis of the influence of nCRT on the extent and nature of intra-tumoral genetic heterogeneity in rectal cancer may provide insights into mechanisms of resistance. Locally advanced rectal cancer patients underwent pre-treatment biopsies. At the time of surgery, tissue from the treated tumor was obtained and analyzed. Pre- and post-treatment specimens were subjected to whole exome and confirmatory deep sequencing for somatic mutations. Copy number variation was assessed using OncoScan SNP arrays. Genomic data were analyzed using PyClone to identify sub-clonal tumor population following nCRT. Alterations that persisted or were enriched in the post-treatment tumor specimen following nCRT were defined for each patient. Thirty-two samples were obtained from ten patients. PyClone identified 2 to 10 genetic sub-clones per tumor. Substantial changes in the proportions of individual sub-clones in pre- versus post-treatment tumor material were found in all patients. Resistant sub-clones recurrently contained mutations in TP53, APC, ABCA13, MUC16, and THSD4. Recurrent copy number variation was observed across multiple chromosome regions after nCRT. Pathway analysis including variant alleles and copy number changes associated with resistant sub-clones revealed significantly altered pathways, especially those linked to the APC and TP53 genes, which were the two most frequently mutated genes. Intra-tumoral heterogeneity is evident in pre-treatment rectal cancer. Following treatment, sub-clonal populations are selectively modified and enrichment of a subset of pre-treatment sub-clones is seen. Further studies are needed to define recurrent alterations at diagnosis that may contribute to resistance to nCRT.