The genetic basis of size variation in the reproductive organs of tomatillo (Physalis philadelphica) is unknown. Here we report that the expression levels of the gene Physalis Organ Size 1 (POS1) are positively associated with size variation in P. philadelphica reproductive organs such flowers, berries and seeds. POS1 knockdown results in smaller flowers and berries with smaller cells as compared with their wild-type counterparts. Conversely, POS1 overexpression promotes organ size without increasing the cell number. The first introns of the POS1 alleles from the large, intermediate and small tomatillo groups contain one, two and three 37-bp repeats, respectively. Furthermore, our results show that copy variation of repeats in the first intron of POS1 alleles results in differential expression of this gene. Thus, co-variation in tomatillo reproductive organ sizes can be attributed to the novel regulatory variation in POS1.