DNA methylation, a key epigenetic modification, influences gene expression, represses transposable elements, and mediates plant responses to environmental stress by adding methyl groups to cytosine residues (5mC) in CG, CHG, and CHH contexts. There are several methods for locus-specific DNA methylation analysis, each with its own advantages and limitations, including bisulfite sequencing PCR (BSP), methylation-specific PCR (MSP), bisulfite pyrosequencing, epityper, methylation-sensitive restriction enzyme (MSRE)-based approaches such as MSRE-Southern blot and MSRE-PCR, methylation-dependent restriction enzyme methods like McrBC, and Affinity Enrichment-based techniques. Here, we present a detailed protocol for locus-specific DNA methylation analysis, highlighting sodium bisulfite sequencing as the gold standard due to its single-base resolution, simplicity, and high accuracy. This protocol utilizes the EZ DNA Methylation-Gold™ Kit for bisulfite conversion, which selectively deaminates unmethylated cytosines to uracil while preserving methylated cytosines. The procedure involves treatment with the CT conversion reagent, followed by thermal cycling and purification using Zymo-Spin™ IC columns. To ensure specific and unbiased amplification of bisulfite-converted DNA, primers are carefully designed using tools such as MethPrimer and BiSearch. Following bisulfite conversion and PCR amplification of the target region, the amplified products are cloned into a binary vector and subjected to Sanger sequencing, enabling single-base resolution analysis of DNA methylation patterns. This approach allows for precise mapping of DNA methylation at promoters, enhancers, and transposable elements, providing valuable insights into gene regulation and phenotypic variation. Locus-specific DNA methylation analysis in plants has wide-ranging applications, including the study of epigenetic responses to stress, transgenerational inheritance, and crop improvement.