Clonal evolution within the tumor microenvironment is central to understanding disease initiation, progression, and therapeutic response and resistance. Resolving these dynamics requires high accuracy spatial transcriptomics that can map driver mutations and cellular phenotypes directly in intact tissue. Direct in Sample Sequencing (DISS) on AVITI24 TM enables the measurement of transcriptome and targeted gene expression directly in intact tissues sections without library preparation, linking genotype and phenotype at high resolution to resolve the complex clonal architecture in the tumor microenvironment. AVITI24 TM utilizes two complementary approaches for spatial profiling with DISS: a 3' transcriptome method using poly-T probes to capture polyadenylated mRNA, and a targeted strategy employing custom probes designed against expressed variants or a panel of hundreds of RNA markers. The AVITI24 TM system automatically performs probe hybridization, cDNA extension, probe circularization, and rolling-circle amplification onboard. DISS chemistry is designed for flexibility, using single-ended probes and supporting up to 100 sequencing cycles with >80% Q30, enabling sensitive detection of a broad range of expressed variants including SNPs, indels, and fusions through long, high quality reads of the transcriptome, not currently capable with dual-sided probe or padlock-based detection methods. To demonstrate variant detection in cancer with DISS, we profiled somatic driver mutations expressed in fresh frozen and FFPE colorectal cancer sections, including multiple hot-spot SNVs in KRAS and TP53, TGFBR2 and APC indels, and ALK gene fusions. Targeted DISS achieved sensitive detection of mutations with variant allele frequency (VAF) down to 5% for genes of diverse expression levels. Utilizing unbi2ased transcriptomic profiling, we accurately resolved major cell types within the tumor microenvironment, with results from sequential tissue sections demonstrating high concordance with RNA seq (R² = 0.7). Spatial mapping of these alterations onto tissue morphology uncovered regional clonal architecture and mutational heterogeneity within intact tumor microenvironments. Using DISS chemistry in tissue, transcriptomics from the same tissue section across a panel of custom genomic targets enabled a highly personalized and sensitive approach to identify both cell types and their associated oncogenic mutations with spatial precision.