Color is an important indicator in evaluating the quality of pearls. Despite the well-developed technology of pearl production, evidence for what determines pearl color is largely absent. In the present study, a 2 × 2 complete double-row implantation experiment was conducted using the purple and white strains of mussels of Hyriopsis cumingii in three different aquaculture environments. The effect of purple and white strains of mussels on seedless pearl color was analyzed using a mixed linear model. The results showed that the effect of the donor mussel, which provides the mantle tissues, on seedless pearl color was significant (P < 0.001), whereas neither the strain of the host mussel nor the aquaculture environment had a significant influence on pearl color. The estimated values for the effect of the donor mussel on pearl color parameters CIE L*, a*, and b* were –9.35, 2.49, and 3.33, respectively. This study provides direct evidence that the color of a seedless pearl is mainly determined by the donor mussel, and both the host mussel and the aquaculture environment have a negligible role in the determination of pearl color.