BACKGROUND: Strawberry (Fragaria spp.) is prone to natural polyploidization. Strawberry shoots regenerated through tissue culture callus may exhibit variations in ploidy. Rapid and accurate ploidy estimation is important for basic research as well as cultivar improvement. OBJECTIVES: Ploidy-measurement methods are well established in strawberry and other plants. The objective of this work was to define the strengths and limitations to various ploidy-estimation tests. METHODS: Measurements were performed on a set of known diploids and polyploids as well as synthetic colchiploids. Comparisons were made using petiole size, stomatal size, leaf dimensions, and pollen diameter and compared to flow cytometry results. RESULTS: Simple methods like leaf proportions and stomatal size may vary greatly based on environmental factors. Pollen measurement proves reliable, but only within tetraploids arising from a single genotype. Measurements made with flow cytometry reliably indicated ploidy and revealed within-plant variation such as mixoploidy. CONCLUSIONS: Methods for measuring ploidy in Fragaria vary in complexity, ease, execution time and precision. This work defines the strengths and limitations of several methods, along with the considerations required for accurate comparisons between genotypes and ploidy levels.