Understanding the mechanisms of drug resistance is important for the fight against malaria and the development of effective new drugs. Antifolate drug resistance is caused by point mutations in dihydrofolate reductase (DHFR) enzyme. With the availability of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing technology, it is possible to specifically edit desired gene locus in Plasmodium malaria parasite’s chromosomal DNA. By using CRISPR/Cas9 gene editing tool, transgenic P. falciparum having the same genetic background but expressing different DHFR mutant enzymes were generated and assessed for their drug resistance profiles. Our results showed that some transgenic parasites do not confer the same level of resistance to antifolate drugs to the lab strain P. falciparum parasites harbouring the corresponding DHFR mutant enzymes. The knowledge obtained on different level of antifolate resistance suggested that other additional enzyme(s) may contribute to antifolate resistance in malaria parasite.