Polyploidy is prevalent in nature. As the fate of duplicated genes becomes more complicated when the encoded proteins function as oligomers, functional investigations into duplicated oligomer-encoding genes in polyploid genomes will facilitate our understanding of how traits are expressed. In this study, we identified GmCHLI1, a gene encoding the I subunit of magnesium (Mg)-chelatase, which functions in hexamers as responsible for the semi-dominant etiolation phenotype in soybean. Four GmCHLI copies derived from two polyploidy events were identified in the soybean genome. Further investigation with regard to expression patterns indicated that these four copies have diverged into two pairs; mutation in the other copy of the pair that includes GmCHLI1 also resulted in a chlorophyll-deficient phenotype. Protein interaction assays showed that these four GmCHLIs can interact with each other, but stronger interactions were found with mutated subunits. The results indicate that, in polyploidy, deficiency in each copy of duplicated oligomer-encoding genes could result in a mutant phenotype due to hetero-oligomer formation, which is different from the model of allelic dosage or functional redundancy. In addition, we interestingly found an increase in isoflavonoids in the heterozygous etiolated plants, which might be useful for improving soybean seed quality. Significance Statement Clarifying how duplicated genes diverge after polyploidy is important for understanding trait determination. Here we show that mutation in either GmCHLI1 or GmCHLI2, encoding the I subunit of magnesium (Mg)-chelatase, results in a chlorophyll-deficient phenotype, because of the formation of hetero-oligomers in polyploidy.