CYCLOIDIEA (CYC) and its homologues have been studied intensively in the model organism Antirrhinum majus and related species regarding their function in controlling floral dorsoventral (adaxial-abaxial) asymmetry, including aborting the adaxial and lateral stamens. This raises the question whether the same mechanism underlies the great morphological diversity of zygomorphy in angiosperms, especially in Lamiales sensu lato, a major clade predominantly with zygomorphic flowers. To address this, we selected a representative in Gesneriaceae, the sister to the remainder of Lamiales s.l., to isolate CYC homologues and further investigate their expression patterns using locus-specific semiquantitative reverse transcriptase polymerase chain reaction. Our results showed that four CYC homologues in Chirita heterotricha differentiated spatially and temporally in expression, in which ChCYC1D was only expressed in the adaxial regions, and transcripts of ChCYC1C were distributed in both the adaxial and lateral regions, while ChCYC2A and ChCYC2B transcripts were only detected in the young inflorescences. ChCYC1C expression in the lateral regions correlated with abortion of the lateral stamens in C. heterotricha hinted at its gain of function, i.e., expanding from the adaxial to the lateral regions in expression. Correlatively, the protein sequences of ChCYC genes exhibited remarkable divergences, in which some lineage-specific amino acids between GCYC1 and GCYC2 in conserved functional domains and two sublineage-specific motifs between GCYC1C and GCYC1D in GCYC1 genes had further been identified. Our results indicated that ChCYC genes had probably undergone an expressional differentiation and specialization in establishing the floral dorsoventral asymmetry in C. heterotricha responding to different selective pressure after gene duplication.