Background Biliary crystal morphology is best described in patients with gallbladder stones, but most patients undergoing bile collection for microscopy have a clinical diagnosis of acalculous gallbladder disease. We investigated the morphology of biliary crystals in such patients. Study Bile was obtained for polarizing microscopy from fresh cholecystectomy specimens of patients with a clinical diagnosis of acalculous or calculous gallbladder disease. Slides for microscopy were prepared by touch contact with bile in freshly opened gallbladder specimens, and following aspiration of gallbladder bile through a 5-French cannula. Results Bile was examined from five patients with a clinical diagnosis of acalculous gallbladder disease and five patients with known gallstones. Needle-like cholesterol crystals predominated in most patients without gallstones, whereas plate-like and dot-like crystals were more common in patients with gallstones. All three crystal types were seen in most patients. Crystal morphology was not affected by aspiration of bile through a 5-French cannula. Conclusions Birefringent needles and dots should be recognized as cholesterol crystals during bile microscopy. These crystal morphologies may predominate in some patients with a clinical diagnosis of acalculous gallbladder disease.