The aim of this study was to determine the chemical variability of Allamanda cathartica flowers collected in different localities of Santa Catarina State, Brazil. Extractive solutions (25 samples) were analyzed by HPLC-DAD for the qualitative and quantitative profiles of the iridoid plumieride, the flavonoid rutin as well as total flavonoid content expressed in rutin. The chemical variability was evaluated by Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA). The samples demonstrated similar chromatographic profiles. In PCA analysis, Araquari samples AC01, AC08 and AC25 did not group with other samples obtained from other locations. In the HCA analysis, it was possible to observe hierarchially similar groups, generating 5 clusters. Group 5 (samples AC01, AC08 and AC25) presented lower similarity compared to the other groups. This dissimilarity can be explained by the amount of metabolites from each collection. Regarding the content of markers, it was observed a variation between 27 to 230 mg/g, 2.7 to 9.5 mg/g and 7.14 to 17.8 mg/g for plumieride, rutin, and total flavonoids, respectively. The samples from Araquari (AC01, AC08, AC25) presented the highest concentrations of chemical markers. Therefore, for reproducibility and standardization of A. cathartica flower extracts, it is important to determine the location and the plant cultivation conditions.