Burkholderia pseudomallei, a bacterium that causes the disease melioidosis, is intrinsically resistant to many antibiotics. First-line antibiotic therapy for treating melioidosis is usually the synthetic β-lactam, ceftazidime (CAZ), as almost all B. pseudomallei strains are susceptible to this drug. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, which can lead to mortality if therapy is not switched to a different drug in a timely manner. Serial B. pseudomallei isolates obtained from an acute Thai melioidosis patient infected by a CAZ susceptible strain, who ultimately succumbed to infection despite being on CAZ therapy for the duration of their infection, were analyzed. Isolates that developed CAZ resistance due to a proline to serine change at position 167 in the β-lactamase PenA were identified. Importantly, these CAZ resistant isolates remained sensitive to the alternative melioidosis treatments; namely, amoxicillin-clavulanate, imipenem, and meropenem. Lastly, real-time polymerase chain reaction-based assays capable of rapidly identifying CAZ resistance in B. pseudomallei isolates at the position 167 mutation site were developed. The ability to rapidly identify the emergence of CAZ resistant B. pseudomallei populations in melioidosis patients will allow timely alterations in treatment strategies, thereby improving patient outcomes for this serious disease.