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We have clarified the microbiological characterization of Gambian traditional fermented milk (FM), "Kosam." Two samples of FM were collected at two regions in The Gambia in 2019. The microbiota of these samples was analyzed by culture-dependent methods and Illumina sequencing. The number of lactic acid bacteria (LAB) in FM from Central River Region (CRR) and Lower River Region (LRR) was 8.27 ± 0.08 log CFU/ml and 7.21 ± 0.09 log CFU/ml, respectively. While no coliforms and Escherichia coli were detected in CRR-FM, LRR-FM contained 5.73 ± 0.17 log CFU/ml of coliforms and 4.82 ± 0.13 log CFU/ml of E. coli. The dominant viable LAB in FM from CRR was Lactobacillus delbrueckii, followed by Streptococcus lutetiensis, while that from LRR was Lactococcus lactis. The metagenomic analysis also revealed that these species were dominant in these Gambian traditional FM. Furthermore, it also revealed the possibility of the presence of pathogens such as Klebsiella spp. This study enhanced the knowledge of Gambian FM and contributed to the elucidation of microbial communities., (© 2022 Japanese Society of Animal Science.)