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Li-Yun Lin,1– 3,* Jian Li,4,* Hui-Ying Huang,5,6 Xue-Yan Liang,5,6 Ting-Ting Jiang,4 Jiang-Tao Chen,7,8 Carlos Salas Ehapo,9 Urbano Monsuy Eyi,9 Yu-Zhong Zheng,1 Guang-Cai Zha,1 Dong-De Xie,7,8 Yu-Ling Wang,7,8 Wei-Zhong Chen,5,6 Xiang-Zhi Liu,5,6 Min Lin1,5,6 1School of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou, Guangdong Province, People’s Republic of China; 2University of Chinese Academy of Sciences, Beijing, People’s Republic of China; 3CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, People’s Republic of China; 4Department of Human Parasitology, School of Basic Medical Sciences; Department of Infectious Diseases, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei Province, People’s Republic of China; 5Department of Medical Laboratory, Chaozhou People’s Hospital Affiliated to Shantou University Medical College, Chaozhou, Guangdong Province, People’s Republic of China; 6Department of Medical Genetics, Shantou University Medical College, Shantou, Guangdong Province, People’s Republic of China; 7The Chinese Medical Aid Team to the Republic of Equatorial Guinea, Guangzhou, Guangdong Province, People’s Republic of China; 8Department of Medical Laboratory, Huizhou Central Hospital, Huizhou, Guangdong Province, People’s Republic of China; 9Department of Medical Laboratory, Malabo Regional Hospital, Malabo, Equatorial Guinea*These authors contributed equally to this workCorrespondence: Min LinSchool of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou, Guangdong Province, People’s Republic of ChinaTel/Fax +86 768-2317422Email konfutea@hotmail.comPurpose: Antimalarial drug resistance is one of the major challenges in global efforts tocontrol and eliminate malaria. In 2006, sulfadoxine-pyrimethamine (SP) replaced with artemisinin-based combination therapy (ACT) on Bioko Island, Equatorial Guinea, in response to increasing SP resistance, which is associated with mutations in the dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes.Patients and Methods: To evaluate the trend of molecular markers associated with SP resistance on Bioko Island from 2011 to 2017, 179 samples collected during active case detection were analysed by PCR and DNA sequencing.Results: Pfdhfr and Pfdhps gene sequences were obtained for 90.5% (162/179) and 77.1% (138/179) of the samples, respectively. For Pfdhfr, 97.5% (158/162), 95.7% (155/162) and 98.1% (159/162) of the samples contained N51I, C59R and S108N mutant alleles, respectively. And Pfdhps S436A, A437G, K540E, A581G, and A613S mutations were observed in 25.4% (35/138), 88.4% (122/138), 5.1% (7/138), 1.4% (2/138), and 7.2% (10/138) of the samples, respectively. Two classes of previously described Pfdhfr-Pfdhps haplotypes associated with SP resistance and their frequencies were identified: partial (IRNI-SGKAA, 59.4%) and full (IRNI-SGEAA, 5.5%) resistance. Although no significant difference was observed in different time periods (p> 0.05), our study confirmed a slowly increasing trend of the frequencies of these SP-resistance markers in Bioko parasites over the 7 years investigated.Conclusion: The findings reveal the general existence of SP-resistance markers on Bioko Island even after the replacement of SP as a first-line treatment for uncomplicated malaria. Continuous molecular monitoring and additional control efforts in the region are urgently needed.Keywords: malaria, drug resistance, dihydrofolate reductase, dihydropteroate synthase, sulfadoxine-pyrimethamine