<p>To investigate the differences in gene expression and functional enrichment associated with anaplasia and laterality in retinoblastoma patients. Two datasets were selected from the GEO database of the National Center for Biotechnology Information of the US, including chip data of gene expression in tumor tissue from 36 patients with retinoblastoma. After grouping by anaplasia or laterality, the data were reanalyzed with Student’s t-test in R and Bioconductor software. The functional enrichment of genes significantly related to anaplasia and laterality was explored via GSEA. The functions of synapse assembly and synaptic signaling were enhanced in clinical patients with severe anaplasia, while the function of photoreceptors was reduced; abnormal conjugate eye movement and lipid metabolic processes were upregulated in unilateral patients. Gene expression may be different in retinoblastoma patients with different anaplasias and lateralities. The consequential difference in gene functional pathways could be associated with clinical symptoms.</p>