This study evaluated molecular techniques in diagnosis and species identification of Malaria parasites in a small endemic location (Bourausso village near Nouna City) versus traditionally used microscopic examination, conducted on 190 of inhabitants of this village, only pregnant women were excluded. By conventional microscopy of Giemsa stained blood smears: prevalence of Plasmodium species, falciparum, malariae and ovale infections was respectively 79.4% (151/190), 3.7% (7/190), 0 %. Also 3.9% (6/152) showed double species infection and 0 % showed triple species infection, while the nested PCR method performed on blood samples spotted on filter paper for the detection of Plasmodium species-specific ssrRNA gene was 95.3 % (181/190), 14.7 %(28/190), and 10 %(19/190). And 18.2 % (33/181), 3.9 %(7/181) double and triple species infection was detected respectively. In surveillance of pfcrt K76T mutation associated with Chloroquine resistance 45.3% of P.falciparum isolates were detected to harbour this mutation. This study showed the need of correct and efficiently informative method for diagnosis of sub-microscopical malaria infection that allow rapid and effective treatment and control.