Background: Libya has been malaria-free since 1973. The risk of malaria re-introduction to Libya is increasing because of the increase in imported malaria cases due to immigration to Libya from countries where malaria is endemic. Cases are mainly due to P. falciparum and Sub-Saharan Africa is the most common origin. This study was conducted to investigate the prevalence of malaria positive cases among illegal immigrants in the southern region of Libya. Methods: A prospective, observational, multi-center study was conducted. Three hundred and three illegal immigrants from 12 different countries were included. Enzyme-linked immunosorbent assay was used to detect the presence of serum malaria antibodies. Results: A total of 303 immigrants were included in the study with mean age of 25.78±5.92 years. Of them, 195 tested positive for malaria antibodies. Two hundred and sixty nine of the participants came from Brak Al-Shati, 16 from Sebha and 18 from Bergan centers, with 175, 15 and 5 positive cases, respectively. Most of the positive cases (172) arrived in Libya within 1-10 months. The highest number of positive cases (70) came from Ghana followed by (40) from Niger and (39) from Bangladesh. Conclusions: Illegal immigration is a major malaria re-introduction route. The issue of illegal immigration have to be treated urgently to stop the huge influx of illegal immigrants and increase the surveillance activities of infectious diseases in order to keep Libyan territories as a malaria-free lands. Key words: Illegal immigration, Malaria, Libya.