This work suggested that in Physalis PFGLO1-PFDEF primarily determined corolla and androecium identity, and acquired a novel role in gynoecia functionality, while PFGLO2-PFTM6 functioned in pollen maturation only. The B-class MADS-box genes play a crucial role in determining the organ identity of the corolla and androecium. Two GLOBOSA-like (GLO-like) PFGLO1 and PFGLO2 and two DEFICIENS-like (DEF-like) PFDEF and PFTM6 genes were present in Physalis floridana. However, the double-layered-lantern1 (doll1) mutant is the result of a single recessive mutation in PFGLO1, hinting a distinct divergent pattern of B-class genes. In this work, we utilized the tobacco rattle virus (TRV)-mediated gene silencing approach to further verify this assumption in P. floridana. Silencing of PFGLO1 or/and PFDEF demonstrated their primary role in determining corolla and androecium identity. However, specific PFGLO2 or/and PFTM6 silencing did not affect any organ identity but showed a reduction in mature pollen. These results suggested that both PFGLO2 and PFTM6 had lost their role in organ identity determination but functioned in pollen maturation. Evaluation of fruit setting in reciprocal crosses suggested that both PFGLO1 and PFDEF might have acquired an essential and novel role in the functionality of gynoecia. Such a divergence of the duplicated GLO-DEF heterodimer genes in floral development is different from the existing observations within Solanaceae. Therefore, our research sheds new light on the functional evolution of the duplicated B-class MADS-box genes in angiosperms.