AbstractA technique that produces large numbers of good quality pachytene chromosome preparations has been developed for Populus. Anthers at the pachytene stage of meiosis are used as materials. There are two main modifications in our method relative to the traditional squashing method that address the challenges the thick cytoplasm observed during the pachytene phase of meiosis presents. One is the temperature during squashing, i.e., the slide is placed on a 52°C heater for squashing. The other is the removal of the cover slip using 45% acetic acid. © 2016 by John Wiley & Sons, Inc.