The digestibility of soybean meal can be severely impacted by trypsin inhibitor (TI), one of the most abundant anti-nutritional factors present in soybean seeds. TI can restrain the function of trypsin, a critical enzyme that breaks down proteins in the digestive tract. Soybean accessions with low TI content have been identified. However, it is challenging to breed the low TI trait into elite cultivars due to a lack of molecular markers associated with low TI traits. We identified Kunitz trypsin inhibitor 1 (KTI1, Gm01g095000) andKTI3(Gm08g341500) as two seed-specific TI genes. Mutantkti1andkti3alleles carrying small deletions or insertions within the gene open reading frames were created in the soybean cultivarGlycine maxcv.Williams 82(WM82) using the CRISPR/Cas9-mediated genome editing approach. The KTI content and TI activity both remarkably reduced inkti1/3mutants compared to theWM82seeds. There was no significant difference in terms of plant growth or maturity days ofkti1/3transgenic andWM82plants in greenhouse condition. We further identified a T1 line, #5-26, that carried double homozygouskti1/3mutant alleles, but not the Cas9 transgene. Based on the sequences ofkti1/3mutant alleles in #5-26, we developed markers to co-select for these mutant alleles by using a gel-electrophoresis-free method. Thekti1/3mutant soybean line and associated selection markers will assist in accelerating the introduction of low TI trait into elite soybean cultivars in the future.