In this study, we correlated the results of concurrent molecular and cytogenetic detection of entity-defining translocations in adults with acute leukemia to determine the frequency of cryptic translocations missed by conventional cytogenetics (CC) and of recurrent, prognostically relevant translocations not detectable by multiplex reverse transcriptase-polymerase chain reaction (MRP). During a 5.5-year period, 442 diagnostic acute leukemia specimens were submitted for MRP-based detection of 7 common recurrent translocations: t(8;21), t(15;17), inv(16), t(9;22), t(12;21), t(4;11), and t(1;19), with a detection rate of 15.2% (67/442). CC was performed in 330 (74.7%) of 442 cases. In 7 of these 330 cases, CC missed the translocation detected by MRP. In 50 additional cases, CC revealed 1 of the MRP-detectable translocations (all were also MRP positive), yielding a false-negative rate of 12% (7/57) for the CC assay. The remaining 140 of 190 cases with clonal cytogenetic changes harbored abnormalities that were not targeted by the MRP assay, including 8 that define specific acute myeloid leukemia entities. This study revealed the frequent occurrence of false-negative, entity-defining CC analysis and highlighted the complementary nature of MRP and CC approaches in detecting genetic abnormalities in acute leukemia.