Background: Rectal cancer is increasing in incidence in young patients, with emerging data demonstrating differences when compared to colon cancer in tumor biology. Additional data demonstrate differences in the genetic basis of colorectal cancer (CRC) based on patient age at diagnosis. This study investigated genomic differences between rectal and colon cancers in the context of patient age. Methods: To determine genomic differences in tumors from younger and older patients with CRC, DNA was extracted from formalin-fixed, paraffin-embedded sections from 4699 cases of CRC, representing 698 cases of rectal cancer and 4001 of colon cancer. Comprehensive genomic profiling was performed on hybridization-captured, adaptor ligation-based libraries to a mean specimen median sequencing depth of 688X for all coding exons and selected introns of up to 403 cancer-related genes. Patients were binned by age. P values were calculated using Fisher’s exact test. Odds ratios were calculated and 95% confidence intervals (95%CI) were calculated using standard methods. Results: FAM123B showed more frequent truncating mutations and deletions in patients under age 40 with rectal cancer as compared to colon cancer (3.7:1 in patients age < 40; p = 0.022). In older age groups this ratio decreased and then reversed (age 40-55: 1.3:1; p = 0.425 age 56-75: 1:1.5; p = 0.136 age > 75: 1:4.3; p = 0.227). Other genes with differential truncation and deletion mutation rates between rectal and colon cancer within age-matched cohorts included SMAD2 (p = 0.031), MLL2 (p = 0.027), ETV6 (p = 0.017), ASXL1 (p = 0.012), and ARID1A (p = 0.057). Gene amplification events were compared between rectal and colon cancers and demonstrated differences in FLT3 (OR = 0.59, 95%CI: 0.39-0.91), JAK2 (OR = 0.12, 95%CI: 0.01-1.06), CD274 (PD-L1) (OR 0.07, 95%CI: 0-0.84), PDCD1LG2 (OR = 0.07, 95%CI: 0-0.84), and PRKC1 (OR = 0.07, 95%CI: 0-0.84). Conclusions: In this analysis, multiple genomic differences were demonstrated between colon and rectal cancers. These lend support to the subdivision of colorectal cancer by anatomic site. These findings also raise possible therapeutic implications of colorectal subsite origin, and warrant further study.