Watermelon (Citrullus lanatus L.) anther and pollen development are regulated by various genes. The gene expression was explored, and differential expression genes (DEGs) related to anther and pollen development were identified by collecting young male floral buds from the pollen tetrad and mature stages of watermelon recessive genetic male sterility lines. Then, cytological study was carried out by using paraffin section experiment, and four sequencing libraries, namely, MFB-A, MFB-B, MSB-a, and MSB-b, were constructed via RNA-seq analysis. The paraffin section experiment indicated the abnormal development of tapetum cell prevented the formation of mature pollen, resulting in male sterility. The RNA-seq result showed that 2111 DEGs, including 1287 downregulated and 824 upregulated DEGs, were found in comparative group 1 (MFB-A/MSB-a), and 3214 DEGs, including 1531 downregulated and 1683 upregulated DEGs, were detected in comparative group 2 (MFB-B/MSB-b). GO enrichment analysis showed that six GO terms, including anther development, anther wall tapetum development, pollen wall assembly, sporopollenin biosynthetic process, pollen development, and pollen exine formation, may be related to watermelon male sterility. qRT-PCR analysis was performed, and the correlation coefficient between RNA-Seq and qRT-PCR was 0.849. These results provided an important data resource for dissecting candidate genes and molecular basis governing male sterility in watermelon.