African swine fever (ASF) is a highly fatal hemorrhagic viral disease of domestic pigs caused by ASF virus (ASFV). Partial amplification of the B646L (p72) gene using PPA1/2 primers followed by Neisseria denitrificans (NdeI) restriction digestion of the PCR products differentiates eastern from southern African ASFV. ASFV circulating between 2010 and 2013 in eastern and southern Tanzania are of southern African origin. A suspected ASF outbreak was reported in northern Tanzania in 2013. Whether the ASFV circulating in eastern and southern Tanzania had spread to northern Tanzania in 2013 has not been determined. The aim of this study was to determine whether the ASFV circulating in eastern and southern Tanzania have genetic similarity to ASFV circulating in northern Tanzania. Partial amplification of the B646L (p72) gene using PPA1/2 primers was performed on ASFV circulating in eastern, southern and northern Tanzania followed by NdeI restriction digestion of the PCR products and nucleotide sequencing. PPA1/2 PCR products of ASFV circulating in eastern and southern Tanzania in 2013 were not cleaved by NdeI restriction endonuclease while those circulating in northern Tanzania were cleaved by NdeI restriction endonuclease. Nucleotide sequencing of PPA1/2 PCR products confirmed the absence of NdeI restriction site in ASFV circulating in eastern and southern Tanzania and its presence in ASFV circulating in northern Tanzania. ASFV in eastern and southern Tanzania is of southern Africa origin while ASFV in northern Tanzania is of eastern Africa origin indicating the co-circulation of two different strains of ASFV in Tanzania in 2013. NdeI restriction digestion can be used to distinguish between the two 2013 ASFV strains without the need for nucleotide sequencing.