HIV-2 surveillance has been carried out in Malaysia for more than 25 years ago. Tests to discriminate HIV-1 and HIV-2 are available but the options of test are limited and the need to develop a new in-house HIV-2 real-time reverse transcription PCR (RT-PCR) is crucial. A study was done on 29 samples from hospitals in Malaysia which were found to be positive screening for HIV-2 antibodies by the commercial Western Blot assay. These samples were further tested by a Western Blot assay that detects specific antibodies to HIV-2. Detection of HIV-2 genome was then performed by using a commercial kit. Fifteen samples were evaluated by using in-house real-time RT-PCR for HIV-2. Ninety-three percent (27/29) of samples have positive results for HIV-2 on HIV-2 Western Blot with only 2 samples showing indeterminate results. All samples showed negative results for HIV-2 genomes by using a PCR commercial kit and the 15 samples that were subjected to our in-house real-time RT-HIV-2 PCR were also tested negative for HIV-2 RNA. Results of HIV-2 Western Blot did not reflect the actual positivity as both HIV-1 and HIV-2 antibodies may cross-react with either viral proteins. None of the samples was confirmed positive for HIV-2 by the commercial and in-house real-time RTPCR. In-house real-time RT-HIV-2 PCR assay can be further used to confirm the presence of HIV-2 genome. Up to the year 2015, Malaysia is still free from HIV-2 infection.