To increase our understanding of the genes involved in flower development in citrus, we identified genes differentially expressed between juveniles and adults of an early-flowering mutant of trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf.) by suppressive subtractive hybridization (SSH) and macroarray hybridization analyses. Based on dot blot analysis, we confirmed that the juvenile subtracted cDNA library comprised genes upregulated during floral induction, inflorescence development and flowering. A total of 190 nonredundant expressed sequence tags were identified that were related flower development. The temporal and spatial expression patterns of four SSH-enriched genes were studied in adult precocious trifoliate orange by real-time PCR. Among these differentially expressed genes, the expression pattern of C5-B16 was closely correlated with inflorescence development and flowering. The full-length cDNA of C5-B16 was isolated by 5' and 3' rapid amplification of cDNA ends. Sequence analysis indicated that C5-B16 is a novel gene in citrus.