Recent reports of haemagglutinin antigen (HA) mismatch between vaccine composition strains and circulating strains, have led to renewed interest in influenza B viruses. Additionally, there are concerns about resistance to neuraminidase inhibitors in new influenza B isolates. To assess the potential impact in Ghana, we characterized the lineages of influenza B viruses that circulated in Ghana between 2016 and 2017 from different regions of the country: Southern, Northern and Central Ghana. Eight representative specimens from the three regions that were positive for influenza B virus by real-time RT-PCR were sequenced and compared to reference genomes from each lineage. A total of eleven amino acids substitutions were detected in the B/Victoria lineage and six in the B/Yamagata lineage. The strains of influenza B viruses were closely related to influenza B/Brisbane/60/2008 and influenza B/Phuket/3073/2013 for the Victoria and Yamagata lineages, respectively. Three main amino acid substitutions (P31S, I117V and R151K) were found in B/Victoria lineages circulating between 2016 and 2017, while one strain of B/Victoria possessed a unique glycosylation site at amino acid position 51 in the HA2 subunit. Two main substitutions (L172Q and M251V) were detected in the HA gene of the B/Yamagata lineage. The U.S. CDC recently reported a deletion sub-group in influenza B virus, but this was not identified among the Ghanaian specimens. Close monitoring of the patterns of influenza B evolution is necessary for the efficient selection of representative viruses for the design and formulation of effective influenza vaccines.