Background: The emergence of Plasmodium falciparum resistance to widely used antimalarial drugs such as chloroquine has made malaria control and treatment much more difficult. In Yemen, 60% of the total population live in malarious areas. The problem of chloroquine resistance in Yemen is gradually worsening since the detection of first indigenous cases of P. falciparum resistance to chloroquine in 1989. Methods: In a cross-sectional malariometric parasitic survey, 447 Yemeni children were enrolled from two selected districts (Hethran and Al-mafatch) representing Taiz Governorate. Duplicate thin and thick blood smears were prepared, stained with Giemsa stain and examined microscopically. Fifty-six students satisfied all criteria of the WHO for the assessment of P. falciparum response to chloroquine using a 7-day in vivo test. Results: Out of 447 examined slides, 83 cases (100%) were found with falciparum malaria. The overall malaria parasite rate in Taiz Governorate was 18.6%, a prevalently mesoendemic condition. The obtained results of the 7-day in vivo study revealed that out of 83 P. falciparum cases who completed the study period, 56 cases did not respond to the standard dose of chloroquine, i.e. the overall resistance rate was 16.1%. The prevalence of chloroquine resistance was higher in the Hethran district (19.4%) compared with 10.0% in the Al-mafatch district. The majority had an RI resistance level. Conclusion: Chloroquine resistance of the local strain of P. falciparum was recorded in all studied districts in Taiz Governorate. This calls for an urgent revision of the current malaria treatment policy which still considers chloroquine as the first-line drug for treatment of uncomplicated P. falciparum malaria. To assess the magnitude of the problem, these districts could be the basis of future sentinel posts for continuous monitoring of chloroquine resistance in the whole country.