Synthetic lethality is thought to play an important role in anticancer therapies. Herein, to understand the potential distributions and relationships between synthetic lethal interactions between genes, especially for pairs deriving from different sources, we performed an integrative analysis of genes at multiple molecular levels. Based on inter-species phylogenetic conservation of synthetic lethal interactions, gene pairs from yeast and humans were analyzed; a total of 37,588 candidate gene pairs containing 7,816 genes were collected. Of these, 49.74% of genes had 2-10 interactions, 22.93% were involved in hallmarks of cancer, and 21.61% were identified as core essential genes. Many genes were shown to have important biological roles via functional enrichment analysis, and 65 were identified as potentially crucial in the pathophysiology of cancer. Gene pairs with dysregulated expression patterns had higher prognostic values. Further screening based on mutation and expression levels showed that remaining gene pairs were mainly derived from human predicted or validated pairs, while most predicted pairs from yeast were filtered from analysis. Genes with synthetic lethality were further analyzed with their interactive microRNAs (miRNAs) at the isomiR level which have been widely studied as negatively regulatory molecules. The miRNA-mRNA interaction network revealed that many synthetic lethal genes contributed to the cell cycle (seven of 12 genes), cancer pathways (five of 12 genes), oocyte meiosis, the p53 signaling pathway, and hallmarks of cancer. Our study contributes to the understanding of synthetic lethal interactions and promotes the application of genetic interactions in further cancer precision medicine.