The aim of the study was to estimate the prevalence of bovine tuberculosis (BTB) and to identify the mycobacterial species causing BTB in a dairy farm and research farm. Six hundred and eighty-five cattle were screened for BTB by using the Comparative intradermal tuberculin test (CTT). Positive reactors were slaughtered and carcasses were taken for isolation of mycobacterial species. This was followed by speciation of isolates using both standard conventional and molecular assays. Seventeen of the cattle were positive by CTT, giving a crude BTB prevalence of 2.48% among cattle from the two farms. Six of the 17 samples (35.30%) yielded positive acid-fast bacilli cultures and three of the isolates were identified as Mycobacterium tuberculosis complex (MTBC), which were sub-divided into two Mycobacterium tuberculosis sensu scrito (Mtb) and one Mycobacterium africanum; the remaining three were Mycobacterium other than tuberculoisis (MOTT). Spoligotyping further characterised the two Mtb isolates as Ghana (spoligotype Data Base 4 number 53) and Latin American Mediterranean (LAM), whilst spoligotyping and Single Nucleotide Polymorphism (SNP) analysis typed the M. africanum as West African 1. Microseq 500 analysis identified two of the MOTT as Mycobacterium flavescens and Mycobacterium Moriokaense respectively, whilst the remaining one could not be identified. This study observed the prevalence of bovine TB among cattle from two farms in Ghana as 2.48% and confirms the public health importance of M. africanum as a pathogen in Ghana.