Objective To validate the application of a sensitive and specific high-throughput DNA microarray chip method for rapid detection of four species of Plasmodium simultaneously. Methods The blood samples with infections of different species of Plasmodium (P. vivax, P. falciparum, P. ovale, P. malariae) identified by microscopy and fluorescent PCR were collected from quarantined passengers at airport, and used for validating the sensitivity and specificity of the high-throughput DNA microarray chip to detect Plasmodium spp.. DNAs were extracted from the blood samples and quantified by the quantitative real-time PCR (RT-PCR). The sensitivity of the DNA microarray was determined by the series dilutions of extracted DNA samples. The specificity of the DNA microarray was determined by cross reacting with the specific Plasmodium sample mixed with the same amount of DNA extracted from P. knowlesi, Cryptosporidium parvum and Balantidium coli. The microarray chip was also used for detecting 50 blood samples collected from patients with suspected malaria from the border area of Yunnan Province, compared with results of microscopy. Results The outcome of the DNA microarray detection of plasmodium spp. completely matched the results detected by microscopy and PCR. The whole procedure only took 2 hours. The chip was able to detect as low as 78 copies/μl of P. vivax, 87 copies/μl of P. malariae, 135 copies/μl of P. falciparum and 302 copies/μl of P. ovale. There was no cross-reaction with P. knowlesi, C. parvum and B. coli. The microarray chip was used to detect 50 blood samples from suspected malaria patients from Yunnan border, 18 of them was identified as infection with P. vivax, 12 for P. falciparum, and 1 for P. malariae. The chip results were totally consistent with the microscopic results. Conclusion The high-throughput DNA microarray is a rapid, sensitive and specific method to detect different species of Plasmodium infections with no cross-reaction to other species of protozoans.