Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9) screening is a simple screening method for locating loci under specific conditions, and it has been utilized in tumor drug resistance research for finding potential drug resistance-associated genes. This screening strategy has significant implications for further treatment of malignancies with acquired drug resistance. In recent years, studies involving genome-wide CRISPR/Cas9 screening have gradually increased. Here we review the recent application of genome-wide CRISPR/Cas9 screening for drug resistance, involving mitogen-activated protein kinase (MAPK) pathway inhibitors, poly (ADP-ribose) polymerase inhibitors (PARPi), alkylating agents, mitotic inhibitors, antimetabolites, immune checkpoint inhibitors (ICIs), and cyclin-dependent kinase inhibitors (CDKI). We summarize drug resistance pathways such as the KEAP1/Nrf2 pathway MAPK pathway, and NF-κB pathway. Also, we analyze the limitations and conditions for the application of genome-wide CRISPR/Cas9 screening techniques.