Background: Malaria is a major public health problem in Africa despite the recent reports on its decline in several parts of the world. Resistance to most conventional antimalarials led to the adoption of artemisinin combination therapy (ACT) as the first-line treatment of uncomplicated malaria in Africa. ACTs are contraindicated in pregnancy due to their side effects. As a result sulphadoxine-pyrimethamine (SP) remained the drug of choice in intermittent preventive treatment in pregnant women (IPTp). A point mutation, K540E of dihydropteroate sythase (dhps) gene of P. falciparum has been observed to act as a single predictor of in vivo SP treatment failure. WHO has recommends SP-IPTp program in places where mutation K540E is less than 50%. SP-IPTp may not be recommended in some parts of Tanzania where already K540E is >50%. Objective: To determine the current prevalence of K540E in Tanzania for proper advice on SP-IPTp implementation. Methodology: A prospective cross-sectional survey involving five regions of Tanzania was done to investigate the prevalence of K540E mutation. PCR-RFLP techniques were used for genotyping. Results: A total of 541 samples were screened. Except for Mtwara Region (that had 65%), the Pfdhps K540E was > 90% in all the regions. The mutation was > 50% in all regions studied. Conclusions: The prevalence of Pfdhps K540E mutation is >50% in Tanzania. Based on WHO criteria that SP-IPTp should not be used when the prevalence of K540E exceeds 50%, there is an urgent need to find an alternative antimalaria drug to SP for IPTp in Tanzania.