Soybean (Glycine max L.) is gaining in importance due to its many uses, including as a food crop and a source of industrial products, among others. Increasing efforts are made to accelerate soybean research and develop new soybean varieties to meet global demands. Soybean research, breeding, identification, and variety protection all rely on precise genomic information. While DNA markers are invaluable tools for these purposes, the older generations, especially those developed before the advent of genome sequencing, lack precision and specificity. Thankfully, advancements in genome sequencing technologies have generated vast amounts of sequence data over the past decade, allowing precise and high-resolution analyses. However, making sense of the genomic information requires a certain level of professional training and computational power, which are not universally available to researchers. To address this, we generated a set of PCR-based DNA markers out of the existing genomic data from 228 popular soybean varieties that offer precise, unambiguous genomic information and can be easily adapted in various applications. A standard operating procedure (SOP) was also designed for these markers and validated on diverse soybean varieties to ensure their reproducibility. This user-friendly universal panel of DNA markers, along with the SOP, will facilitate soybean research and breeding programs through simple applications.