Accurate determination of ploidy level of putative polyploid plants is essential for tree breeding and other applications. Methods for ploidy determination include quantification of chromosome numbers in root-tip cells via light microscopy and indirect assessment via anatomical and morphological traits. Flow cytometry is potentially a high-throughput method to quantify nuclear DNA content; however, it does not allow determining chromosome numbers and interfering compounds often prevent its use. Microscopy-based quantification of chromosomes in active root-tip cells remains the most unambiguous method for ploidy determination, although root tips are difficult to obtain from field-grown plants, and light microscopy can result in insufficient resolution in species with many and small chromosomes. Here, we present a robust technique that uses 2, 4-diamidino-2-phenylindole (DAPI) dye and 1000-fold magnification fluorescence microscopy for quantification of chromosomes in root and shoot tips of woody angiosperms and gymnosperms, and overcomes the reported difficulties. Rather than the conventional tip squashing, spreading tips on glass slides resulted in very good chromosome separation in diverse species, with up to 56 chromosomes and a chromosome size of 2–20 μm. Chromosome counts were performed in diploid Agathis robusta, Elaeocarpus angustifolius, Eucalyptus robusta, Paulownia tomentosa, Pongamia pinnata and Toona ciliata, and di- and tetraploid Acacia crassicarpa and Citrus species.