Melioidosis, a disease endemic to northern Australia and Southeast Asia, is caused by the soil saprophyte Burkholderia pseudomallei. The indirect hemagglutination assay (IHA) is the most frequently used serological test to help confirm exposure to the causative organism. However, despite culture-confirmed disease, patients often have a negative IHA result at presentation and occasionally fail to seroconvert in serial testing. We retrospectively examined results for all patients with culture-confirmed melioidosis from our laboratory between January 1996 and August 2008. One hundred forty patients had a recorded IHA titer at presentation, 71 of which were positive at a titer of 1:40 or greater. Fifty-three patients went on to have subsequent IHAs 1 month or more after presentation. The relationships between IHA responses and clinical features were examined. The presence of bacteremia was significantly associated with a negative IHA at presentation. The coexistence of diabetes was associated with the presence of a positive IHA at presentation. In total, 14 patients (26%) demonstrated persistently negative IHA titers upon serial testing. No clinical factors were found to be significantly associated with this phenomenon. Supplementary testing using melioidosis-specific immunoglobulin G by EIA demonstrated different effects, with only Aboriginal or Torres Straits Islander ethnicity being significantly associated with a positive EIA at presentation. Reasons for these findings are examined, and directions for future research are discussed.