Analyzing protein-protein interaction (PPI) networks has been a crucial prerequisite for understanding the molecular basis for most of the diseases. Although several investigations have been carried out on PPI network analysis, none of them explicitly considered binding affinity as a criterion for the analysis. In this work, we have performed a systematic analysis of protein-protein interaction networks in five organisms based on the binding affinity of interacting partners. We observed that eukaryotes are marginally dominated with high affinity complexes and an opposite trend was observed in prokaryotes. In addition, hub-hub interactions have the highest percentage of “high affinity” interactions followed by hubnonhub and nonhub-nonhub interactions. Further, all organisms contain hubs, which are enriched specifically with high or low affinity complexes irrespective of the dominance of these interactions. Sub network analysis indicates that the closed triad motifs with high and low affinity complexes are more significant than the open motifs. The analysis of clustering coefficient and amino acid properties showed specific preferences in different organisms. These findings deepen the knowledge of PPI networks and provide useful insights for target identification in drug discovery.