Radial spokes are ubiquitous components of motile cilia and flagella and play an essential role in transmitting signals that regulate the activity of the dynein motors, and thus ciliary and flagellar motility. In some organisms the 96 nm axonemal repeat unit contains only a pair of two spokes, RS1 and RS2, while most organisms have spoke triplets with an additional spoke RS3. The spoke pair in Chlamydomonas flagella has been well characterized, while spoke triplets have received less attention. Here, we used cryo-electron tomography and subtomogram averaging to visualize the 3D structure of spoke triplets in Strongylocentrotus purpuratus (sea urchin) sperm flagella in unprecedented detail. Only small differences were observed between RS1 and RS2, but the structure of RS3 was surprisingly unique and structurally different from the other two spokes. We observed novel doublet specific features that connect RS2, RS3 and the nexin-dynein regulatory complex, three key ciliary and flagellar structures. The distribution of these doublet specific structures suggests that they could be important for establishing the asymmetry of dynein activity required for the oscillatory movement of cilia and flagella. Surprisingly, a comparison with other organisms demonstrated both that this considerable radial spoke heterogeneity is conserved and that organisms with radial spoke pairs contain the basal part of RS3. This conserved radial spoke heterogeneity may also reflect functional differences between the spokes and their involvement in regulating ciliary and flagellar motility.