Background: Malaria is a major public health problem in the Sudan including Gezira State. The genetic diversity of malaria parasite may affect its transmission model and control strategies. Merozoite Surface Protein-2 (MSP-2) is a glycoprotein expressed on the surface of merozoites that has been considered as one of the candidates for blood stage malaria vaccines. The MSP-2 gene is located on chromosome 2 and is composed of five blocks the most polymorphic of which is the central block 3. The gene is encoded by highly divergent alleles, grouped into two dimorphic families FC27 and IC/3D7. Methods: Filter paper blood spot samples (89) of P. falciparum isolates were collected from the children attending Wad Medani Pediatric Teaching Hospital, Gezira State, Sudan from September 2022 to February 2023. Parasite density was estimated per microliter using thick blood film. The genomic DNA was extracted, and the MSP-2 gene was genotyped by nested PCR using allele-specific primers for P. falciparum. Results: For the MSP-2 gene, the most frequent allele was the FC27 (68.5%) allelic family in the study area compared to IC1/3D7 (59.6%) allelic family, and samples positive for the both alleles IC1/3D7 and FC27 were (32 and 35.9% respectively). The total multiplicity of infection (MOI) of MSP-22 was (1.48). 14.61% of studied samples were found to have multi-clonal isolates and the mixed infections were 40.45%. In regarding to parasitemia, the level 1 – 5000 parasite/µl were (58.9%) for IC1/3D7 and (70.0%) for FC27. The mixed infections represent (75%) and the MOI was 1.58. On the other hand, in the parasitemia level >10000 parasite/µl there were (62.1%) for IC1/3D7 and (65.6%) for FC27. The mixed infections represent (65.5%) and the MOI was (1.27). Conclusion: The study found highly genetic polymorphisms with diverse allele types of MSP-2 as well as the high MOI of P. falciparum isolates in Gezira State, Sudan.