There is variation in the responsiveness of locally advanced rectal cancer to neoadjuvant chemoradiation, from complete response to total resistance. This study compared genetic variation in rectal cancer patients who had a complete response to chemoradiation versus poor response, using tumor tissue samples sequenced with genomics analysis software. Rectal cancer patients treated with chemoradiation and proctectomy June 2006-March 2017 were grouped based on response to chemoradiation: those with no residual tumor after surgery (CR, complete responders, AJCC-CPR tumor grade 0, n = 8), and those with poor response (PR, AJCC-CPR tumor grade two or three on surgical resection, n = 8). We identified 195 variants in 83 genes in tissue specimens implicated in colorectal cancer biopathways. PR patients showed mutations in four genes not mutated in complete responders: KDM6A, ABL1, DAXX-ZBTB22, and KRAS. Ten genes were mutated only in the CR group, including ARID1A, PMS2, JAK1, CREBBP, MTOR, RB1, PRKAR1A, FBXW7, ATM C11orf65, and KMT2D, with specific discriminating variants noted in DMNT3A, KDM6A, MTOR, APC, and TP53. Although conclusions may be limited by small sample size in this pilot study, we identified multiple genetic variations in tumor DNA from rectal cancer patients who are poor responders to neoadjuvant chemoradiation, compared to complete responders.© 2020 Taylor & Francis Group, LLC.