Citrus suavissima 'seedless' (Wuzi Ougan), a bud variant of C. suavissima (Ougan), maintaining almost all excellent characteristics of 'Ougan' except for seedlessness, and chalcone synthase (CHS), playing an important role on plants by providing floral organs with different types of flavones, were used in this study. To indicate the role of the CHS gene family in male sterility in Wuzi Ougan, four CHS homologs were differentially expressed in a comparative analysis of transcriptome and proteome between Wuzi Ougan and Ougan. The CHS homologs in Wuzi Ougan and Ougan were isolated and their expressions were individually analyzed in different developmental stages of anthers and floral organs. Then, a bioinformatics analysis of the CHS gene family was carried out based on the clementine mandarin genome. Results showed that the nucleotide sequences of CHS homologs derived from Wuzi Ougan and Ougan shared more than 98% similarity with their homologs in clementine mandarin. Real-time polymerase chain reaction (PCR) showed that Ciclev10005133m, Ciclev10030093m, and Ciclev10015535m were down-regulated at microsporcyte in Wuzi Ougan; whereas, Ciclev10005133m, Ciclev10001405m, and Ciclev10015535m were down-regulated in Wuzi Ougan at meiosis. In terms of tetrad, Ciclev10030093m and Ciclev10001405m were down-regulated in Wuzi Ougan (P < 0.05). After pollen maturation, Ciclev10001405m and Ciclev10005133m were mainly expressed at anthers (P < 0.05). The bioinformatics analysis revealed 13 CHS genes that were identified in clementine mandarin. In conclusion, members of the CHS family maintained high similarity in coding sequence (CDS) and were differentially expressed between Wuzi Ougan and Ougan in anther development, which was expected to contribute to male sterility in Wuzi Ougan.