Background: Biomarkers are a key tool in early detection, prognostication, survival, and predicting treatment response of colorectal cancer (CRC). However, little is known about biomarker testing for CRC patients in real-life clinical practice in China. This study aimed to address the usage of biomarker testing and analyze factors related to its acceptance among Chinese patients with advanced CRC. Methods: A multicenter, cross-sectional, hospital-based clinical epidemiology study was conducted from March 2020 to March 2021. Nineteen hospitals were selected in seven geographical regions of China using stratified, multistage, nonrandomized cluster sampling. Data on demographics and clinical characteristics of each eligible CRC patient in stage III or IV diseases were recorded based on the patients' self-reporting and/ or medical records. In addition, information on whether biomarker testing [RAS, BRAF, and microsatellite instability (MSI)] was performed, the results and timing for performing biomarker testing, and the reasons for refusing biomarker testing were also recorded. Univariate and multivariate logistic regression were conducted to explore the potential factors of biomarker testing. Results: A total of 4,526 patients were enrolled in the study, of whom 41.4%, 36.1%, and 28.2% underwent RAS, BRAF, and MSI testing, respectively. RAS, BRAF, and high-level MSI (MSI-high) mutation rates in Chinese patients with advanced CRC were 37.0%, 9.9%, and 8.1%, respectively. The logistic regression analysis revealed that the treating hospital, age at diagnosis, education, family income, tumor site, history of chemotherapy and radiotherapy, and metastases were dependent factors affecting the utilization of biomarker testing in advanced CRC in China (P<0.005). Conclusions: The biomarker testing rate, especially MSI testing, is less prevalent in clinical practice for patients with advanced CRC in China. Our findings may guide the formulation of biomarker testing of CRC strategies in China and other low-income countries.