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© van de Kooij et al. Human NimA-related kinases (Neks) have multiple mitotic and non-mitotic functions, but few substrates are known. We systematically determined the phosphorylation-site motifs for the entire Nek kinase family, except for Nek11. While all Nek kinases strongly select for hydrophobic residues in the -3 position, the family separates into four distinct groups based on specificity for a serine versus threonine phospho-acceptor, and preference for basic or acidic residues in other positions. Unlike Nek1-Nek9, Nek10 is a dual-specificity kinase that efficiently phosphorylates itself and peptide substrates on serine and tyrosine, and its activity is enhanced by tyrosine auto-phosphorylation. Nek10 dual-specificity depends on residues in the HRD+2 and APE- 4 positions that are uncommon in either serine/threonine or tyrosine kinases. Finally, we show that the phosphorylation-site motifs for the mitotic kinases Nek6, Nek7 and Nek9 are essentially identical to that of their upstream activator Plk1, suggesting that Nek6/7/9 function as phosphomotif amplifiers of Plk1 signaling., Dutch Cancer Society (Grant BUIT 2015-7546), National Cancer Institute (U.S.) (Grant K99CA226396), National Institutes of Health (U.S.) (Grant 01-GM104047), National Institutes of Health (U.S.) (Grant 01-ES015339), National Institutes of Health (U.S.) (Grant R35-ES028374), National Cancer Institute (U.S.) (Grant P30-CA14051), National Institute of Environmental Health Sciences (Grant P30-ES002109)