Colorectal cancer, a malignant gastrointestinal tumor with high morbidity and mortality, has long been plagued by difficulties in treatment and high recurrence rates, seriously threatening patients’ lives and health. With the continuous development of molecular biology technology, targeted therapy has emerged and shown great value in improving the survival and prognosis of patients with colorectal cancer. However, drug resistance is common in clinical practice, which directly impairs therapeutic efficacy. Next-Generation Sequencing (NGS), a high-efficiency and high-throughput molecular detection technology, can deeply analyze the genomic characteristics of tumors, accurately identify relevant molecular targets, and provide important support for the study of drug resistance mechanisms and the formulation of individualized countermeasures. This paper discusses the drug resistance mechanisms of targeted therapy for colorectal cancer based on NGS and the corresponding countermeasures, aiming to provide reference for relevant practitioners.