| Sumario: | <i>Fasciola gigantica</i> is an important trematode that affects the health of animals and humans in tropical and subtropical countries, including Malawi. Information on the genetic diversity and population structure of <i>F. gigantica</i> is important to understanding the parasite's transmission patterns/ and in monitoring the development of resistance to commonly used anthelmintic agents. This study aimed to analyze the genetic diversity and population structure of <i>Fasciola</i> species collected from cattle at slaughter slabs and abattoirs in selected districts of Malawi. A total of 27 adult liver flukes were collected from cattle at slaughter slabs and abattoirs in the northern region (n = 12), central region (n = 5), and southern region (n = 10). The mitochondrial cytochrome c oxidase I (COI) gene and nicotinamide adenine dinucleotide dehydrogenase 1 (ND1) gene were amplified and the amplicons were sequenced for all samples. The sequences obtained were used to investigate genetic diversity through median-joining networks and phylogenetic analysis. Tajima’s D test and Fu’s Fs statistics were used to determine the population structure. Based on the analyzed COI and ND1 sequences, all samples were identified as <i>F. gigantica</i>. Single nucleotide polymorphisms (SNPs) were identified at 18 and 17 positions for COI and ND1 genes, resulting in 10 and 5 haplotypes, respectively. The haplotype diversities were 0.867 and 0.479 for COI and ND1 gene sequences, respectively. The population genetic structure indices showed a population that has undergone a recent expansion. This study provides baseline epidemiological data on the genetic diversity and population structure of <i>F. gigantica</i> in Malawi; which is important for its control.
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