The goal of the study was to compare the performance of a fluorescence-based multiplex PCR fragment analysis to a direct sequencing method for detecting CEBPA mutations in patients with acute myeloid leukemia. Thirty-three samples were selected from a larger study of 107 cases of acute myeloid leukemia by screening for CEBPA mutations by sequence analysis. Of ten identified mutations, six (insertions and deletions) were detected by both sequencing and fragment methods. The fragment analysis method did not detect the remaining four base substitutions because the method cannot detect changes that result in identically sized products. The multiplex PCR fragment length analysis method therefore failed to detect substitution mutations accounting for 40% of total CEBPA mutations in our patient set. Our results indicate that fragment length analysis should not be used in isolation, and that direct sequencing is required to evaluate CEBPA gene mutational status in acute myeloid leukemia. A combination of the two assays may offer some advantages, chiefly in permitting more sensitive detection by fragment length analysis of insertions and deletions.