Accumulating evidence suggests that colorectal cancer (CRC) should be viewed as a heterogeneous disease, with proximal and distal CRCs showing multiple biological and clinical differences. The aim of this study was to develop a clinicopathological, molecular and protein profile for CRCs based on their region and thus providing insight into their heterogeneity. CRC patients (n=399) were evaluated for clinicopathologic and molecular features including K-RAS, BRAF and MSI status. Tumors were also screened for expression of 50 immunohistochemical markers linked to major signaling pathways involved in tumor-progression or immune response. Proximally located tumors show significantly larger tumor size, higher T-stage, higher tumor grade and more frequent mucinous histologic subtype compared to the distal colon and rectum. The frequency of BRAF mutation and MSI-high phenotype were significantly higher in proximal colon cancers. There is a significant difference in regional expression of 10 tumor-associated markers (CDX2, CD44v6, CD44s, TOPK, nuclear beta-catenin, pERK, APAF-1, E-cadherin, p21 and bcl2) and 4 immune response markers (CD68, CD163, FoxP3 and TIA-1). In multivariate analysis CD44s, CD44v6, nuclear beta-catenin and CD68 expression was found to best discriminate left- versus right-sided colon cancers. Tumor diameter, pT stage and MSI status best distinguish right-sided colon cancers from rectal cancers and pT stage and E-cadherin best discriminate left-sided colon cancers and rectal cancers. These data along with existing evidence for the presence of distinct regional embryological origin and gene expression profile are highly supportive of the concept that proximal and distal CRCs are distinct clinicopathologic entities.