The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y₁-S₂-P₃-T₄-S₅-P₆-S₇. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y₁, S₂, T₄, S₅, and S₇ in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S₂ phosphorylation levels within the CTD.