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This study presents the results of a Europe-wide training and Quality Control (QC) exercise carried out within the framework of the European Aerobiology Society’s QC Working Group and European COST Action FA1203 entitled “sustainable management of Ambrosia artemisiifolia in Europe (SMARTER)” with the aim of ensuring that pollen counters in Europe are confident in the identification of Ambrosia pollen grains. A total of 69 analysts from 20 countries examined a test slide by light microscopy, which contained Ambrosia pollen and pollen from other Asteraceae that could be recorded in the atmosphere at the same time of year (i.e. Artemisia, Iva, and Xanthium). Daily average pollen concentrations produced by individual participants were compared with the assigned value and the bias was measured by z-score. Both the assigned value and standard deviation for proficiency testing were calculated following the consensus value principle (ISO13528:2005) from the results reported by all the participants in the test. It took a total of 531 days from when the exercise commenced until all 69 analysts reported their results. The most outliers were reported for Artemisia pollen concentrations followed by Xanthium and Iva. The poor results for Artemisia and Xanthium were probably caused by low concentrations on the test slide leading to larger bias due to the unequal distribution of pollen over the microscope slide. Participants performed the best in identifying and quantifying Ambrosia pollen. Performing inter-laboratory ring tests with the same sample is very time consuming and might not be appropriate for large-scale proficiency testing in aerobiology. Pollen with similar morphology should be included in the education process of aerobiologists.