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Background  Malaria is a leading public health problem in Ethiopia. Accurate diagnosis of  Plasmodium  infections is crucial for the reduction of malaria in tropical areas and for epidemiological studies. The role of light microscopy (LM) as gold standard has been questioned and, therefore, new molecular methods have been developed for the detection of  Plasmodium  species. The aim of the present work was to compare different malaria diagnostic methods in order to detect the most common species of  Plasmodium  and to broaden the knowledge of malaria prevalence in a hospital in a rural area in Ethiopia.    Methods  A cross-sectional survey of 471 individuals was carried out in a hospital in the rural area of Gambo (Ethiopia). Blood samples were prepared for microscopic observation and collected in filter paper for Seminested-Multiplex PCR (SnM-PCR) and real time PCR (qPCR) testing. The SnM-PCR was considered as the gold standard technique and compared with the rest. Thus, agreement between SnM-PCR and LM was determined by calculating Kappa Statistics and correlation between LM and qPCR quantification was calculated by pair-wise correlation co-efficient.    Results  Samples analysed by LM and SnM-PCR were positive for  Plasmodium  sp. 5.5% and 10.5%, respectively. Sensitivity was 52.2% by LM and 70% by qPCR. Correlation co-efficient between microscopy counts and qPCR densities for  Plasmodium vivax  was R 2  = 0.586. Prevalence was estimated at 7% (95% CI: 4.7–9.3).  Plasmodium vivax  was the dominant species detected and the difference was statistically significant ( χ  2  = 5.121  p  < 0.05). The highest prevalence of the parasite (10.9%) was observed in age groups under 15 years old.    Conclusion  Accurate malaria diagnostic methods have a great effect in the reduction of the number of malaria-infected individuals. SnM-PCR detection of malaria parasites may be a very useful complement to microscopic examination in order to obtain the real prevalence of each  Plasmodium  species. Although SnM-PCR shows that it is a good tool for the determination of  Plasmodium  species, today light microscopy remains the only viabletool for malaria diagnosis in developing countries. Therefore, re-inforcement in the training of microscopists is essential for making the correct diagnosis of malaria.  Plasmodium vivax  was the predominant species in Gambo, a meso-endemic area for this species.

Microscopy and molecular biology for the diagnosis and evaluation of malaria in a hospital in a rural area of Ethiopia