622 Background: Recent reports have demonstrated inferior outcomes for patients with right-sided colorectal cancer (CRC) compared to patients with left-sided disease (Schrag et al 2016, JCO 34 (suppl; abstr 3505)) as well as differences in treatment response based on disease sidedness (Venook et al 2016, JCO 34 (suppl; abstr 3504)). However, the biological and genetic underpinnings of these clinical differences are incompletely understood. Methods: We compared mutation rates among 38 genes in a retrospective review of next-generation sequencing data of CRC samples obtained in routine clinical practice at a single academic medical center. Primary location was identified via chart review. Right = cecum to transverse colon; Left = descending colon to rectum. Results: Among 293 samples (167 left-sided, 103 right, 23 synchronous or without clear primary), BRAF and CTNNB1 mutations were more prevalent in right-sided CRC. BRAF was mutated in 15.5% of right-sided CRC (95% CI: 8.5-22.5%) compared to 4.8% (CI: 1.6-8.0%) (p=0.003). CTNNB1 was mutated in 3.9% of right-sided CRC (CI: 0.2-7.6%) compared to no instances of CTNNB1 mutations in left-sided disease (p=0.01). Among right-sided CRC, there was a trend toward more KRAS mutations at 57.3% (CI: 47.7-66.8%) versus 44.9% (CI: 37.4-52.5%) and more PIK3CA mutations at 26.2% (CI:17.7-34.7%) versus 17.4% (CI: 11.6-23.1%), though these differences did not rise to the level of statistical significance. The overall rate of BRAF mutations in our sample (8.9%, CI: 5.5-12.3%) was consistent with the rate of BRAF mutations among large intestine adenocarcinomas in the COSMIC database (10.6%, CI: 10.4-10.8%), lending support to the external validity of these data. Conclusions: These differing mutation rates may implicate these genetic pathways in the mechanisms underlying the discrepant outcomes and treatment responses between right and left-sided disease described in prior studies. Further work is needed to more clearly elucidate genetic difference between right and left-sided CRC, as well as the mechanistic relationship between these mutations and prognosis.