Plant male sterility (MS) is an important agronomic trait that provides an efficient tool for hybridization and heterosis utilization of crops. Based on phenotypic and cytological observations, our study performed a multi-comparison transcriptome analysis strategy on multiple sterile and fertile rubber tree varieties using RNA-seq. Compared with the male-fertile varieties, a total of 1590 differentially expressed genes (DEGs) were detected in male-sterile varieties, including 970 up-regulated and 620 down-regulated transcripts in sterile varieties. Key DEGs were further assessed focusing on anther development, microsporogenesis and plant hormone metabolism. Twenty DEGs were selected randomly to validate transcriptome data using quantitative real-time PCR (qRT-PCR). Eleven key genes were subjected to expression pattern analysis using qRT-PCR and fluorescence in situ hybridization. Among them, nine genes, i.e., A6, GAI1, ACA7, TKPR1, CYP704B1, XTH26, MS1, MS35 and MYB33, that regulate callose metabolism, pollen wall formation, tapetum and microspores development were identified as candidate male-sterile genes. These findings provide insights into the molecular mechanism of male sterility in rubber tree.