BACKGROUND In 2002, the World Health Organization recommended that artemisinin-based combination therapy (ACT) be used to treat uncomplicated malaria. Dihydroartemisinin-piperaquine and artesunate-pyronaridine are two of these new combinations. The aim of the present work was to assess the distribution of the in vitro values of pyronaridine (PND) and piperaquine (PPQ) and to define a cut-off for reduced susceptibility for the two anti-malarial drugs. METHODS The distribution and range of the 50% inhibitory concentration values (IC₅₀) of PND and PPQ were determined for 313 isolates obtained between 2008 and 2012 from patients hospitalized in France for imported malaria. The statistical Bayesian analysis was designed to answer the specific question of whether Plasmodium falciparum has different phenotypes of susceptibility to PND and PPQ. RESULTS The PND IC₅₀ values ranged from 0.6 to 84.6 nM, with a geometric mean of 21.1 ± 16.0 nM (standard deviation). These values were classified into three components. The PPQ IC₅₀ values ranged from 9.8 to 217.3 nM, and the geometric mean was 58.0 ± 34.5 nM. All 313 PPQ values were classified into four components. Isolates with IC₅₀ values greater than 60 nM or four-fold greater than 3D7 IC₅₀ are considered isolates that have reduced susceptibility to PND and those with IC₅₀ values greater than 135 nM or 2.3-fold greater than 3D7 IC₅₀ are considered isolates that have reduced susceptibility to PPQ. CONCLUSIONS The existence of at least three phenotypes for PND and four phenotypes for PPQ was demonstrated. Based on the cut-off values, 18 isolates (5.8%) and 13 isolates (4.2%) demonstrated reduced susceptibility to PND and PPQ, respectively.