The JIL-1 kinase localizes to interband regions of Drosophila polytene chromosomes and phosphorylates histone H3 Ser10. Analysis of JIL-1 hypomorphic alleles demonstrated that reduced levels of JIL-1 protein lead to global changes in polytene chromatin structure. Here we have performed a detailed ultrastructural and cytological analysis of the defects in JIL-1 mutant chromosomes. We show that all autosomes and the female X chromosome are similarly affected, whereas the defects in the male X chromosome are qualitatively different. In polytene autosomes, loss of JIL-1 leads to misalignment of interband chromatin fibrils and to increased ectopic contacts between nonhomologous regions. Furthermore, there is an abnormal coiling of the chromosomes with an intermixing of euchromatic regions and the compacted chromatin characteristic of banded regions. In contrast, coiling of the male X polytene chromosome was not observed. Instead, the shortening of the male X chromosome appeared to be caused by increased dispersal of the chromatin into a diffuse network without any discernable banded regions. To account for the observed phenotypes we propose a model in which JIL-1 functions to establish or maintain the parallel alignment of interband chromosome fibrils as well as to repress the formation of contacts and intermingling of nonhomologous chromatid regions.