Background: Colorectal cancer (CRC) remains one of the leading causes of morbidity and mortality around the world. Familial cancer syndromes are common in CRC, inherited syndromes including Lynch syndrome (LS), familial adenomatous polyposis (FAP), and MutY human homolog (MUTYH)-associated polyposis (MAP) were well defined. But still, there are not well-established evidence between other several genes and CRC risk. Expanded multi-gene testing may be an alternative to find out the underlying mechanism for genetic susceptibility. Our study aims to assess the germline alternation landscape of Chinese CRC patients. Methods: We performed hybrid capture-based next-generation sequencing (NGS) of 381 genes on tissues from patients with CRC between January 01, 2017 and December 02, 2019 in 3D Medicines database. Genomic alterations including single nucleotide variation (SNV), insertions/deletions, copy number variations, gene rearrangement and fusions were assessed. Results: 1360 CRC patients were included for analysis, in which 95 (7.0%) patients harbored pathogenic (80/95) and likely pathogenic (15/95) germline mutations in 28 cancer predisposition genes. 33 subjects had pathogenic variants associated with Lynch syndrome (15 mutations in MLH1, 9 mutations in MSH2, 6 mutations in MSH6, 3 mutations in PMS2, respectively). 8 subjects had pathogenic variants associated with FAP (3 APC), MAP (3 MUTYH) and Li-Fraumeni syndrome (2 TP53). In addition, 14 pathogenic mutations in moderate penetrance genes (5 ATM, 3 BLM and 6 CHEK2) were found. The frequency of pathogenic BRCA mutations were 1 alternation in BRCA1 and 3 alternations in BRCA2. Conclusions: 7.0 % of patients with Chinese CRC carried germline cancer susceptibility gene mutations. Multi-gene testing would provide references for assessing genetic susceptibility and managing increased surveillance.