Autotetraploid rice has greater genetic variation and higher vigor than diploid rice, but low pollen fertility is one of the major reasons for low yield of autotetraploid rice. Very little is known about the molecular mechanisms of low pollen fertility of autotetraploid rice. In this study, cytological observations and microarray analysis were used to assess the genetic variation during pollen development in autotetraploid and diploid rice. Many abnormal chromosome behaviors, such as mutivalents, lagged chromosomes, asynchronous cell division, and so on, were found during meiosis in autotetraploid. Microsporogenesis and microgametogenesis in autotetraploid rice was similar to diploid rice, but many different kinds of abnormalities, including microspores degeneration, multi-aperture, and abnormal cell walls, were found in autotetraploid rice. Compared with diploid rice, a total of 1,251 genes were differentially expressed in autotetraploid rice in pollen transcriptome, among them 1,011 and 240 genes were up-regulated and down-regulated, respectively. 124 and 6 genes were co-up-regulated and co-down-regulated during three pollen development stages, respectively. These results suggest that polyploidy induced up-regulation for most of the genes during pollen development. Quantitative RT-PCR was done to validate 12 differentially expressed genes selected from functional categories based on the gene ontology analysis. These stably expressed genes not only related to the pollen development genes, but also involved in cell metabolism, cell physiology, binding, catalytic activity, molecular transducer activity, and transcription regulator activity. The present study suggests that differential expression of some key genes may lead to complex gene regulation and abnormal pollen development in autotetraploid rice.