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Large and distinct families of receptor-like kinases (RLKs) play elemental roles in many fundamental processes of plants. The proline-rich extensin-like receptor kinase (<i>PERK</i>) family is one of the most pivotal classes of RLKs. To date, there have been no comprehensive or published studies conducted on the <i>PERK</i> gene family in <i>Glycine max</i>. This research aimed to characterize the role of the <i>PERK</i> gene family in cultivated soybean using a systematic array of bioinformatic and experimental approaches. We identified 16 <i>PERK</i> members in <i>G</i>. <i>max</i> through local BLASTp, using <i>PERK</i> members from <i>Arabidopsis thaliana</i> as a query. Tissue expression of genes, predicted via tissue specific expression analysis from the soybean database “SoyBase”, revealed that these <i>PERK</i> genes exhibit differentiated expression patterns in various plant organs. The gene structure was predicted via Gene Structure Display Server (GSDS). Phylogeny was demonstrated through an evolutionary tree employing the neighbor-joining method. Subcellular localization of proteins was identified via “Softberry” and <i>cis</i>-acting elements were identified through PlantCARE. The KASP (Kompetitive Allele Specific PCR (KASP)) marker was developed for the <i>GmPERK-1-C</i> and <i>GmPERK-1-T</i> allele, targeting position 167 nt in the CDS region. Genotyping results indicated that <i>GmPERK-1</i> exhibits promising potential for utilization in molecular breeding programs for soybean to increase crop yield. Collectively, our findings indicate that <i>G. max</i> accessions harboring the <i>GmPERK-1-C</i> allele exhibit significantly higher thousand grain weight compared to accessions carrying the <i>GmPERK-1-T</i> allele. This research enhances the understanding of the molecular roles of <i>PERK</i> genes in <i>G. max</i>, providing valuable insights for the utilization of favorable genetic variations in soybean molecular breeding programs.