Background: Lemur tail kinase 3 (LMTK3) plays a critical role in multiple cellular pathways such as Wnt signaling, KIT modulation, and the estrogen receptor pathway. We previously reported that LMTK3 gene polymorphisms are associated with clinical outcome in patients with CRC, and that LMTK3 and estrogen-mediated signaling play a crucial role in CRC tumorigenesis in vitro. Here, we aimed to characterize the molecular features associated with LMTK3 gene expression in CRC. Methods: 20,219 CRC were tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (592 genes or WES) and RNA (WTS). Top quartile transcripts per million (TPM) for LMTK3 expression (Q4) were considered high while bottom quartile (Q1) low. Consensus molecular subtypes (CMS) were assessed using RNAseq. Cell infiltration (CI) in the tumor microenvironment (TME) was estimated by QuantiSEQ. X² and Fisher-Exact tests were used, and significance was determined as p-value adjusted for multiple comparisons (q< 0.05). Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy (interferon gamma [IFNγ] score; T-cell inflamed signature [TIS] score). Results:LMTK3 expression was highest in left-sided (0.98 median TPM) followed by right-sided (0.96) and lowest in rectal tumors (0.92). No difference in patient sex distribution was observed between Q1 and Q4 cohorts. Overall, LMTK3 Q1 was associated with TMB-high (≥ 10 Mut/Mb, 11.4 vs 8.7%) and dMMR/MSI-H (8.1 vs 4.6%) (q< 0.0001); however, these associations were no longer significant in pMMR/MSS tumors. In the pMMR/MSS cohort, LMTK3 expression was highest in CMS4 (1.09 median TPM) followed by CMS2 (0.98), CMS1 (0.88), and lowest in CMS3 (0.70, all intergroup q< 0.05). LMTK3 high showed differential rates of mutations and copy number alterations (CNA) in several genes, including higher mutation rates of APC, TP53, and CDX2 CNA, while lower mutation rates of KMT2A/D, ASXL1, ATM, SMAD4, RNF43 and AMER1 in the overall cohort (q< 0.05). In the pMMR/MSS cohort, the associations with APC, TP53, SMAD4 and ATM mutations and CDX2 CNA still held true (q< 0.05). High LMTK3 was associated with higher immune CI in the TME, including B cells, neutrophils, NK cells, CD4+ T cells, monocytes and dendritic cells, while macrophages and Tregs were lower regardless of tumor MSI status (fold change: 0.83-2.4, all q< 0.0001). The IFNγ score was significantly lower in LMTK3 Q4 while the TIS score was significantly higher in LMTK3 Q4 (q< 0.0001). Conclusions: Our data show a strong association between LMTK3 gene expression and distinct molecular features and TME immune cell infiltration in CRC. These findings suggest that LMTK3 may be an important molecular factor that plays a role in determining the composition of the TME, thus targeting LMTK3 could represent a novel strategy in selected CRC subgroups.