Background: One of the major challenges in developing an effective vaccine against asexual stages of Plasmodium falciparum is genetic polymorphism within parasite population. Understanding the genetic polymorphism like block 2 region of merozoite surface protein (msp-1) genes of P. falciparum enlighten mechanisms underlining disease pathology, identification of the parasite clone profile from the isolates, transmission intensity and potential deficiencies of the ongoing malaria control and elimination effort in the locality. Detailed understanding of local genetic polymorphism is an input to pave the way for better management, control and elimination of malaria. The aim of this study was to detect the most frequent allelic variant of the merozoite surface protein (msp-1) gene of P. falciparum clinical isolates from selected health facilities in Adama town and its surroundings, Oromia, Ethiopia.Methods: A total of 139 clinical isolates were successfully amplified for msp-1 gene using specific sets of primer. Nested PCR amplification conducted, using specific primers targeting K1, MAD20, and R033 alleles followed by gel electrophoresis for fragment analysis. Based on the detection of a PCR fragment, infections were classified as monoclonal or multiple infections.Result: 19 different size polymorphism of msp-1 gene were identified in the study, with 67(48 %) MAD20, 18 (13 %) K-1 and 18 (13 %) RO33 allelic family. Whereas, the multiple infections were 21(15 %), 8(5.8 %), 4(2.9 %), 3(2.2 %) for MAD20+K-1, MAD20+RO33, K-1+ RO33, and MAD20+K-1, RO33, respectively. The overall Multiplicity of Infection (MOI) was 1.3 and the expected heterozygosity (He) was 0.58 indicating intermediate falciparum malaria transmission.Conclusion: The status of msp-1 allele size polymorphism, MOI and He observed in the study revealed an intermediate genetic diversity of P. falciparum clinical isolates, indicating that the ongoing malaria control and elimination effort should be intensified to effectively monitor the potential malaria resurgence in the study area. Moreover, deriving force that led to high predominance of MAD20 allelic variant revealed in such malaria declining region demands further research.