The phosphorylation states of RNA polymerase II coordinate the process of eukaryotic transcription by recruitment of transcription regulators. The individual residues of the repetitive heptad of the C-terminal domain (CTD) of the biggest subunit of RNA polymerase II are phosphorylated temporally at different stages of transcription. Intriguingly, despite similar flanking residues, phosphorylation of Ser2 and Ser5 in CTD heptads play dramatically different roles. The mechanism of how the kinases place phosphorylation on the correct serine is not well understood. In this paper, we use biochemical assays, mass spectrometry, molecular modeling, and structural analysis to understand the structural elements determining which serine of the CTD heptad is subject to phosphorylation. We identified three motifs in the activation/P+1 loops differentiating the intrinsic specificity of CTD in various CTD kinases. We characterized the enzyme specificity of the CTD kinases-CDK7 as Ser5-specific, Erk2 with dual specificity for Ser2 and Ser5, and Dyrk1a as a Ser2-specific kinase. We also show that the specificities of kinases are malleable and can be modified by incorporating mutations in their activation/P+1 loops that alter the interactions of the three motifs. Our results provide an important clue to the understanding of post-translational modification of RNA polymerase II temporally during active transcription.