Chinese hamster ovary (CHO) cells have been widely used for production of recombinant proteins and therapeutic antibodies. However, owing to the instability and heterogeneity of CHO cells, the development of stable and high-expression recombinant CHO cell lines is often time-consuming. To investigate the mechanisms associated with heterogeneity in protein productivity, we performed transcriptome analysis on the subclones derived from a stable parental CHO clone. Two high-expression subclones and one low-expression subclone were selected based on their similar genomic background and subjected to RNA-seq analysis. Over 100 differentially expressed genes were identified between the subclones with high and low productivity. The molecular functions of the differentially expressed genes were enriched for translational elongation, sterol biosynthetic process, and regulation of secretion. In addition, analyses of the two subclones with high protein expression levels identified over 300 differentially expressed genes involved in DNA metabolic processes, cellular macromolecule catabolic processes, cell cycle, protein catabolic processes, and RNA processing and transcription. A subset of the differentially expressed genes was overexpressed in CHO cells to identify their effects on protein production. Together, these results indicate that transcriptome variation can cause significant inter-cellular heterogeneity in CHO cells and a better understanding of the molecular mechanism underlying heterogeneity might help to improve the production of recombinant proteins by CHO cells.