India is the largest producer and exporter of buffalo (Bubalus bubalis) meat in the world. But there are restrictions on the slaughter of cattle (Bos indicus/Bos taurus) and export of beef (cattle meat) from the country. This led to the incidences of misrepresentation of beef and other low cost meat as buffalo meat during export and hence necessitates reliable techniques for species authentication of buffalo meat. Tests need to be rapid to ensure that export consignments are not held for a long time while meat samples are being authenticated. In the present study, a rapid assay for authentication of buffalo meat was developed by employing alkaline lysis (AL) method of DNA extraction and loop-mediated isothermal amplification (LAMP) using novel primers designed to target mitochondrial D loop region. The results of the AL-LAMP assay were visualized by SYBR Green I dye. Further, the LAMP amplification was confirmed by agarose gel electrophoresis and fluorometry. The developed AL-LAMP assay was validated using raw, heat treated, and admixed/adulterated buffalo meat samples. The AL-LAMP assay could be carried out with minimum instrumentation and hence, holds potential for use as on-site assay for species authentication of buffalo meat.