The mammalian cell cycle is coordinated by primarily four cyclin-dependent kinases (CDKs), which are activated by a family of cyclin proteins to phosphorylate diverse protein effectors of cell growth and division. A wealth of qualitative protein interaction studies have supported a model in which different CDKs have specific cognate cyclin partners. However, there have been few quantitative measurements of binding kinetics and affinity to support our understanding of CDK-cyclin preferences and the structural origins of those preferences. We used a biolayer interferometry (BLI) assay to quantify association and dissociation rates and to determine binding constants for all pairings of the cell-cycle CDKs and cyclins. We found that the highest affinity interactions, including CDK1 for CycB, CDK2 for CycA and CycE, and CDK4 for CycD, involve complexes that are considered canonical and have most often been reported. Structural modeling and mutagenesis experiments demonstrate that specific sequence differences can explain preferential interactions in the case of CDK2 binding to CycA compared to CycD. Finally, we show that all the cell-cycle CDK-cyclin complexes are competent to catalyze ATP phophotransfer with only a few outliers demonstrating relatively high or low catalytic efficiency. The implications of these observations for the potential activation of noncanonical CDK-cyclin pairs in cancer cell proliferation are discussed.