Chronic lymphocytic leukemia (CLL) is the most common leukemia affecting adults in the western world. The clinical course of CLL is highly variable: cases that express mutated immunoglobulin heavy chain variable regions (IgV(H)) typically have a more indolent clinical course compared with those with unmutated IgV(H). The use of the V(H)3-21 variable region has also been found to confer a poor prognosis, independent of mutation status. Here we describe an assay for the identification of the expressed V(H) segment and its mutation status in CLL. This test uses whole blood-derived RNA and PCR primers annealing to the leader regions and the joining region segments. This approach allows more accurate determination of the IgV(H) mutation status relative to using framework region specific V(H) primers. An additional primer specific for the leader region of the V(H)3-21 segment is described and is shown to be necessary to identify this diagnostically important variable region. We successfully analyzed 99 of 103 samples, including five expressing the V(H)3-21 variable region. Approximately 5% of cases had complement determining region 3 sequences similar to previously reported cases, and overrepresentation of the V(H)1-69 segment was observed among unmutated cases. These results confirm the proper functioning and high success rate of this valuable prognostic for CLL designed for the use in a clinical laboratory setting.