Heterosis is the superior performance of F1 hybrids relative to their parental lines for a wide range of traits. In this study, expression profiling and heterosis associated genes were analyzed by RNA sequencing (RNA-Seq) in seedlings of the maize hybrid An’nong 591 and its parental lines under control and heat stress conditions. Through performing nine pairwise comparisons, the maximum number of differentially expressed genes (DEGs) was detected between the two parental lines, and the minimum number was identified between the F1 hybrid and the paternal lines under both conditions, which suggested greater genetic contribution of the paternal line to heat stress tolerance. Gene Ontology (GO) enrichment analysis of the 4518 common DEGs indicated that GO terms associated with diverse stress responses and photosynthesis were highly overrepresented in the 76 significant terms of the biological process category. A total of 3970 and 7653 genes exhibited nonadditive expression under control and heat stress, respectively. Among these genes, 2253 (56.8%) genes overlapped, suggesting that nonadditive genes tend to be conserved in expression. In addition, 5400 nonadditive genes were found to be specific for heat stress condition, and further GO analysis indicated that terms associated with stress responses were significantly overrepresented, and 60 genes were assigned to the GO term response to heat. Pathway enrichment analysis indicated that 113 genes were involved in spliceosome metabolic pathways. Nineteen of the 33 overlapping genes assigned to the GO term response to heat showed significantly higher number of alternative splicing (AS) events under heat stress than under control conditions, suggesting that AS of these genes play an important role in response to heat stress. This study reveals the transcriptomic divergence of the maize F1 hybrid and its parental lines under control and heat stress conditions, and provides insight into the underlying molecular mechanisms of heterosis and the response to heat stress in maize.