We developed a method for preparing male chromosomes from sea urchin hybrid andromerogones created with cryopreserved sperm. We obtained hybrid andromerogones by heterospermic insemination of Hemicentrotus pulcherrimus non-nucleate egg fragments produced by centrifuging unfertilized eggs in a stepwise saccharose density gradient. The hybrid andromerogones showed cleavage rates of 1%-93%, cleaved successively into two- and four- blastomeres and developed to early blastulae. The morulae or early blastulae were treated with colchicine (0.1-1.0 mg/ml), dissociated into single blastomeres by pippeting, swollen with 7%-10% sodium citrate for 10 min and fixed with methanol:acetic acid (3:1). The fixed cells were dropped on slides and air-dried. The andromerogones for 5 sperm species showed a half of their respective diploid chromosome numbers without chromosome elimination. This method is applicable for analysis of the haploid male chromosome complement in sea urchin species for which only sperm can be obtained.