In human medicine, PCR-amplification of the complementarity determining region 3 of the immunoglobulin heavy chain genes followed by polyacrylamide gel electrophoresis (PAGE) is an accepted method to assess clonality in B-cell lymphomas and thereby facilitates the differentiation of neoplasias from benign hyperplasias or reactive infiltrates. To generate a basis for the development of a PCR-based assay for the assessment of clonality in feline B-cell lymphomas we analyzed 28 transcripts (cDNA) of the feline immunoglobulin heavy chain variable region genes (IGHV). Transcripts were generated using techniques for the amplification of unknown sequences (i.e. the SMART RACE and the CapFishing technique) as well as primers derived from sequences of the NCBI Trace archive of the cat. Analysis of this archive revealed traces similar to the human IGHV-1 and IGHV-3 subgroups of genes. By identification of the subgroup-specific leader sequence within the traces, two subgroup-specific primers for this region were designed and used to amplify the heavy chain variable region genes. Using all amplification techniques, transcripts of both subgroups were created and the subgroups were denominated according to their human counterparts as feline IGHV-1 and feline IGHV-3. By aligning previously described transcripts of the feline IGHV genes to our transcripts we were able to assign these to the IGHV-3 subgroup; therefore, this study provides the first description of the feline IGHV-1 subgroup of genes. On the basis of the IGHV-1 and IGHV-3 transcripts we developed a PCR-based assay. For each of the two subgroups we used one sense primer derived from the first and one sense primer derived from the third framework region each in combination with a mixture of three antisense primers derived from the fourth framework region. With these four sets of primers, the assay was able to detect monoclonality in 7/10 (70%) cats with histologically and immunohistochemically diagnosed B-cell lymphomas. In two of these cases, monoclonal rearrangement of the IGHV genes was only detectable with IGHV-1 subgroup-specific primers. Amplification of feline hyperplastic lymphatic tissue only gave results indicative of polyclonal populations. The use of a PCR-based assay in combination with standard techniques for the diagnosis of feline lymphoma is helpful and the characterization of the additional subgroup of feline variable regions genes puts this assay on a broader basis.