Chikungunya (CHIK) virus is enzootic in many countries in Asia and throughout tropical Africa. In Asia the virus is transmitted from primates to humans almost exclusively by Aedes aegypti, while various aedine mosquito species are responsible for human infections in Africa. The clinical picture is characterized by a sudden onset of fever, rash and severe pain in the joints which may persist in a small proportion of cases. Although not listed as a haemorrhagic fever virus, illness caused by CHIK virus can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms. Thus, laboratory confirmation of suspected cases is required to launch control measures during an epidemic. CHIK virus diagnosis based on virus isolation is very sensitive, yet requires at least a week in conjunction with virus identification using monovalent sera. We developed a reverse transcription-polymerase chain reaction (RT-PCR) assay which amplifies a 427-bp fragment of the E2 gene. Specificity was confirmed by testing representative strains of all known alphavirus species. To verify further the viral origin of the amplicon and to enhance sensitivity, a nested PCR was performed subsequently. This RT-PCR/nested PCR combination was able to amplify a CHIK virus-specific 172-bp amplicon from a sample containing as few as 10 genome equivalents. This assay was successfully applied to four CHIK virus isolates from Asia and Africa as well as to a vaccine strain developed by USAMRIID. Our method can be completed in less than two working days and may serve as a sensitive alternative in CHIK virus diagnosis.