Soybean [Glycine max (L.)] is a major source of protein for human and animal feed. Inter- and intra-genotype variation of soybean protein has been investigated by soybean researchers. However, limited sample amount of soybean single seed there is no report that investigated intra-plant variation of soybean protein within soybean plant. Recently a non-destructive NIR (near-infrared reflectance) spectroscopy using single seed grain to analyze seed protein was developed. The objectives of this study were to understand variation of seed protein content within plant and to determine the amount of minimum sample size which can represent protein content for a soybean plant. Frequency distribution of protein content within plant showed normal distribution. There was an intra-cultivar variation for protein content in soybean cultivar Seonnogkong. Difference of protein content among single plants of Seonnokong was recognized at 5% level. Seeds in lower position on plant stem tended to accumulate more protein than in higher position. There was significant difference for protein content between sample size 5 seeds and sample size of more than 5 seeds (10, 20, 30, 40, and 50 seeds) at a soybean plant with 57 seeds however no difference was recognized among sample size (5, 10, 20, and 30 seeds) at a soybean plant with 33 seeds. Around 20% seeds of soybean from single plant needed to determine the protein content to represent protein content of single soybean plant. This study is the first one to report evidence of intra-plant variation for proteincontent which detected by non-destructive NIR spectroscopy using single seed grain in soybean.