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Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder caused by several distinct defects in genes responsible for ciliary beating leading to defective mucociliary clearance often associated with randomization of left/right body asymmetry. PCD individuals with defective radial spoke heads are difficult to diagnose due to lack of gross ultrastructural defects and absence of situs inversus . Thus far, most radial spoke mutations identified are loss-of function mutations, and missense variants have been rarely described. Using immunofluorescence analyses (IF), we studied the consequences of different RSPH9 , RSPH4A , and RSPH1 mutations on the assembly of the radial spoke complex to improve diagnostics in PCD. We identified bi-allelic mutations in RSPH9, RSPH4A and RSPH1 including 7 novel mutations comprising missense variants in 21 PCD individuals (16 families). We performed IF using antibodies directed against the radial spoke head components RSPH9, RSPH1 and RSPH4A to analyzed respiratory cilia of these patients. Absence of RSPH4A due to mutations in RSPH4A results in deficient axonemal assembly of the radial spoke head components RSPH1 and RSPH9. RSPH1 mutant cilia, lacking RSPH1 fail to assemble RSPH9, while RSPH9 mutations result in axonemal absence of RSPH9 but do not affect the assembly of the other head proteins RSPH1 and RSPH4A. Interestingly, our results were identical in individuals carrying loss-of-function mutations, missense variants or one amino acid deletion. Thus, we conclude that Immunofluorescence analysis can improve diagnosis of PCD in patients with loss-of-function mutations as well as missense variants.