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Simple Summary: Cas9 is limited by protospacer adjacent motif (PAM) sequences during the application process, which is particularly evident in insect genome editing. Researchers have discovered Cas9 variants with a wider range of recognition due to the development of genome-editing technology. However, the application of these variants in insects has been ineffective and time-consuming. To tackle this issue, this study established a high-throughput assay system capable of rapidly detecting recognizable PAM sequences of different Cas9 variants in Bombyx mori and the corresponding editing efficiency of the sites. We utilized this system to evaluate the editing ability of two protein variants, xCas9 and Cas9-NG, in Bombyx mori cells. Based on the editing ability of these two proteins, the editable range of the Bombyx mori genome can be greatly expanded, accelerating research on the genome's function. This system is also applicable to other insects for detecting the editing ability of Cas9 variants, thus accelerating the study of silkworm genome function. Genome editing provides novel opportunities for the precise genome engineering of diverse organisms. Significant progress has been made in the development of genome-editing tools for Bombyx mori (B. mori) in recent years. Among these, CRISPR/Cas9, which is currently the most commonly used system in lepidopteran insects, recognizes NGG protospacer adjacent motif (PAM) sequences within the target locus. However, Cas9 lacks the ability to target all gene loci in B. mori, indicating the need for Cas9 variants with a larger editing range. In this study, we developed a high-throughput screening platform to validate Cas9 variants at all possible recognizable and editable PAM sites for target sequences in B. mori. This platform enabled us to identify PAM sites that can be recognized by both xCas9 3.7 and SpCas9-NG variants in B. mori and to assess their editing efficiency. Cas9 shows PAM sites every 13 base pairs in the genome, whereas xCas9 3.7 and SpCas9-NG have an average distance of 3.4 and 3.6 base pairs, respectively, between two specific targeting sites. Combining the two Cas9 variants could significantly expand the targeting range of the genome, accelerate research on the B. mori genome, and extend the high-throughput rapid screening platform to other insects, particularly those lacking suitable NGG PAM sequences. [ABSTRACT FROM AUTHOR]