Soluble acid invertase [SAI (Enzyme Commission 220.127.116.11)] plays an important role in catalyzing the hydrolysis of sucrose into hexoses and regulates floral development. Full-length cDNAs encoding RhSAI1 and RhSAI2 isoforms were cloned from Rhododendron hybrid ‘Yuqilin’ and they exhibited high amino acid sequence identity (89%) to each other. The protein sequences contain highly conserved motifs present in all SAIs, including the β-fructosidase motif N-D-P-(D/N), a putative active site W-E-C-(I/V)-D, and R-D-P. The expression of RhSAI1 and RhSAI2 genes was under spatial and temporal control. Expression of both RhSAI1 and RhSAI2 genes was most abundant in stems, and expression was lowest in roots and leaves, respectively. The expression of RhSAI2 was significantly lower than that of RhSAI1 in all organs. During floral development, RhSAI1 was highly expressed at the earliest stage (Stage I), decreased until Stage III, and increased again at the terminal stage. The pattern of RhSAI2 expression was distinctly different, showing a continuous increase during floral development. Consistent with the levels of RhSAI1 expression, SAI activity decreased during floral development and was inversely correlated with the soluble sugar content. Abundant expression of RhSAI1 at the transcriptional level in addition to high SAI activity during the initial stages of floral development may play a vital role in supplying the energy needed for rapid cell division and growth of flowers.