Objectives: To investigate the predictive accuracy of using a combination of the high pressure liquid chromatography (HPLC) retention time and the relative isoelectric focusing (IEF) position to diagnose rare hemoglobin variants. Methods: A selected group of 40 patients with a rare beta-chain variant were assigned a presumed diagnosis following HPLC and IEF screening and then the variant identified in each case by DNA analysis. The study was conducted at the National Hemoglobinopathy Reference Laboratory, Oxford, United Kingdom, from August 2008 to October 2008. Results: Thirteen out of 14 different variants were predicted accurately in 39 (97.5%) cases, compared to only one each for HPLC and IEF when used individually. A novel amplification refractory mutation system-polymerase chain reaction test was developed for Hb J-Baltimore and used successfully, to provide a simple, rapid, and inexpensive diagnosis. Conclusion: The use of both HPLC retention time and isoelectric focusing position provides an accurate presumed diagnosis of a rare hemoglobin variant in the majority of cases. Amplification refractory mutation system-polymerase chain reaction test can provide a simple, rapid and inexpensive molecular diagnostic method for rare beta-chain variants.