Colorectal cancer (CRC) progression occurs through three stages: adenoma (pre-cancerous lesion), carcinoma in situ (CIS) and adenocarcinoma, with tumor stage playing a pivotal role in the prognosis and treatment outcomes. Despite therapeutic advancements, the lack of stage-specific biomarkers hinders the development of accurate diagnostic tools and effective therapeutic strategies. This study aims to identify stage-specific gene expression profiles and key molecular mechanisms in CRC providing insights into molecular alterations across disease progression. Our methodological approach integrates the use of absolute gene set enrichment analysis (absGSEA) on formalin-fixed paraffin-embedded (FFPE)-derived transcriptomic data, combined with large-scale clinical validation and experimental confirmation. A comparative whole transcriptomic analysis (RNA-seq) was performed on FFPE samples including adenoma ( = 10), carcinoma in situ (CIS) ( = 8) and adenocarcinoma ( = 11) samples. Using absGSEA, we identified significant cellular pathways and putative molecular biomarkers associated with each stage of CRC progression. Key findings were then validated in a large independent CRC patient cohort ( = 1926), with survival analysis conducted from 1336 patients to assess the prognostic relevance of the candidate biomarkers. The key differentially expressed genes were experimentally validated using real-time PCR (RT-qPCR). Pathway analysis revealed that in CIS, apoptotic processes and Wnt signaling pathways were more prominent than in adenoma samples, while in adenocarcinoma, transcriptional co-regulatory mechanisms and protein kinase activity, which are critical for tumor growth and metastasis, were significantly enriched compared to adenoma. Additionally, extracellular matrix organization pathways were significantly enriched in adenocarcinoma compared to CIS. Distinct gene signatures were identified across CRC stages that differentiate between adenoma, CIS and adenocarcinoma. In adenoma, , and were overexpressed, suggesting their involvement in early tumorigenesis, whereas in CIS, and were overexpressed, suggesting their involvement in the transition from benign to malignant stage. In adenocarcinoma, , , and were overexpressed, suggesting their involvement in advanced disease progression. Functional analysis confirmed that and /2 were associated with early tumor development, while and were involved in extracellular matrix remodeling and transcriptional regulation, respectively. Experimental validation with RT-qPCR confirmed the differential expression of the candidate biomarkers (, , , and across the three CRC stages reinforcing their potential as stage-specific biomarkers in CRC progression. These findings provide a foundation to distinguish between the CRC stages and for the development of accurate stage-specific diagnostic and prognostic biomarkers, which helps in the development of more effective therapeutic strategies for CRC.