Maize endosperm accounts for more than 80% of the grain weight. Cell division and grain filling are the two key stages for endosperm development. Previous studies showed that gene expression during differential stages in endosperm development is greatly different. However, information on systematic identification and characterization of the differentially expressed genes between the two stages are limited. In this study, suppression subtractive hybridization (SSH) was used to generate four subtracted cDNA libraries for the two stages using two maize inbreds with large and small grain. Totally, 4,784 differentially expressed sequence tags (ESTs) were sequenced and 902 were non-redundant, which consisted of 344 unique ESTs. Among them 192 had high sequence similarity to the GenBank entries and represent diverse of functional categories, such as metabolism, cell growth/division, transcription, signal transduction, protein destination/storage, protein synthesis and others. The expression patterns of 75.7% SSH-derived cDNAs were confirmed by reverse Northern blot and semi-quantitative reverse transcription polymerase chain reaction, and exhibited the similar results (75.0%). Genes differentially expressed between two key stages for the two inbreds were involved in diverse physiological process pathway, which might be responsible for the formation of grain weight. 43.8% (70 of the 160 unique ESTs) of the identified ESTs were assigned to 39 chromosome bins distributed over all ten maize chromosomes. Eleven ESTs were found to co-localize with previous detected QTLs for grain weight, which might be considered as the candidate genes of grain weight for further study.