With the increasing focus on whole-genome sequencing and genomic studies in non-model organisms, there is a growing demand for reliable and accessible methods to estimate eukaryotic genome sizes across diverse taxa. Accurate genome size determination is essential for advancing genomic research and breeding programs, particularly in plants. Here, we have provided the details of two methodologies used for estimating genome size in clusterbean (Cyamopsis tetragonoloba) flow cytometry [estimated genome size 580.9 Mb ± 0.02 (1C)] and the K-mer based approach (estimated genome 543.2 Mb). Flow cytometry involves staining clusterbean nuclei with a DNA-binding dye and measuring fluorescence intensity using a flow cytometer and provides a rapid genome size estimate by comparing the fluorescence data of unknown samples to a standard reference species with a known genome size. In contrast, the K-mer-based approach employs high-throughput sequencing data to analyze K-mer frequencies in short DNA sequences of length K, enabling genome size estimation through computational models. While this method is highly precise and does not require a reference genome, it necessitates extensive sequencing and bioinformatics analysis. Our study found that flow cytometry offered a quick estimation with some variability, whereas the K-mer based approach yielded a more accurate and detailed genome size estimate, and the fraction of heterozygosity in clusterbean genome was estimated to be in the range of 0.70–0.71%. This comparative analysis highlights the strengths and limitations of each method, assisting researchers in choosing the most suitable technique based on their specific needs and available resources.