To identify the chromosomal loci of allelic loss in intrahepatic cholangiocarcinoma (ICC), we performed an allelotype study of 36 ICCs using 55 genome-wide microsatellite markers. Loss of heterozygosity was found most frequently on 8p (65.6%), 17p (64.7%), and 9p (64.5%), followed by 18q (54.2%), 1p (48.5%), 3p (44.8%), 9q (42.1%), 14q (41.7%), 6q (41.7%), and 1q (40.6%). The fractional allelic loss (FAL) values ranged from 0 to 0.731 (mean, 0.322). Analysis of the relationship between FAL values and clinicopathologic parameters disclosed significantly higher FAL values in moderately to poorly differentiated ICCs than in well-differentiated ones (P < .05). In summary, this study defined for the first time the overall number of chromosomes having allelic loss and the chromosomal arms and/or regions potentially involved in the development of ICC.