Lymphoplasmacytic lymphoma (LPL), marginal zone lymphoma (MZL), and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) are well-defined clinicopathologic entities. However, distinguishing LPL from MZL and from atypical cases of CLL can sometimes be difficult because of overlapping features. Recent studies have identified a recurrent L265P mutation in the MYD88 gene in most cases of LPL. Although this represents a promising diagnostic marker for LPL, the mutation is also reported in rare cases of MZL and CLL (as well as other types of B-cell lymphoma). Detection rates for this mutation have varied depending on the analytic methodology.To assess the diagnostic utility of MYD88 L265P mutation in diagnosing low-grade B-cell lymphomas.We developed a novel pyrosequencing assay for the MYD88 L265P mutation and assessed its diagnostic utility in 317 cases of low-grade B-cell lymphoma (45 LPL [14%], 53 MZL [17%], and 219 CLL [69%]). We incorporated formal clinical and pathologic review of selected cases to ensure the most accurate diagnosis and subclassification.The MYD88 L265P mutation was identified in 43 cases of LPL (96%), including 3 nonimmunoglobulin-M LPL cases. In contrast, the mutation was present in only 2 cases of MZL (4%), and 5 cases of CLL (2%). Thus, pyrosequencing for the MYD88 L265P mutation demonstrates a high clinical sensitivity and specificity to distinguish LPL from MZL and CLL.This study confirms the strong association of the MYD88 L265P mutation with LPL, as well as the existence of rare cases of small B-cell lymphoma that complicate this association.