Abstract: 【Objective】This study aimed to optimize grape chromosome preparation technique and analyze the rDNA distribution characteristics in 14 grape varieties，providing reference for grape chromosome identification，variation analysis，and analysis of cytogenetic background differentiation among grape varieties.【Method】The root tips of 14 grape varieties from hydroponically grown semi-lignified shoots were used as experimental materials. A control group treating root tips with an ice-water mixture for 24 h，followed by 1 MPa nitrous oxide（N₂O）treatment for 30 min was set. The root tips were soaked in 0.2 μmol/L amiprophos-methyl solution（APM）for 2 h，and treated with 1 MPa N₂O for different durations（0，30，and 60 min）. Chromosome preparation conditions were screened，and the proportion of metaphase splitting phases in good shape under each treatment was calculated. The chromosome preparation effects of enzymatic digestion and dropping method and the flame-drying method were compared. The distribution characteristics of rDNA on chromosomes of 14 grape varieties were analyzed using dual-color fluorescence in situ hybridization（FISH） technique with 45S rDNA and 5S rDNA as probes.【Result】The proportion of metaphase splitting phases in good shape in grape root tip materials was only 32.22% in the control group. After FISH，background signals were easily generated and the signals were blurred. The highest proportion of metaphase splitting phases in good shape（80.00%）in chromosome slides was obtained by treating root tips with APM for 2 h and with 1 MPa N₂O for 30 min. The chromosome concentration was appropriate，and the quality of FISH signals was good. The flame-drying method resulted in moderate cell dispersion and a high number of metaphase splitting phases. 45S rDNA and 5S rDNA signals were consistently linked on grape chromosomes. The number of 5S rDNA signals was correlated with the ploidy level，with 2，3 and 4 signals observed in diploid，triploid and tetraploid grapes respectively. The number of 45S rDNA signals was 4，6 and 7 in diploid，triploid and tetraploid grapes respectively，except for Hongxiangjiao grape，which had only 3 signals. The 5S rDNA signals were located on chromosome 17，while the 45S rDNA signals were located on chromosomes 17 and 15. Exceptions were observed in Italy grape and Bixiang seedless grape with two 45S rDNA signals unlinked to 5S rDNA signals located on chromosomes 15 and 16 in Italy grape，and one 45S rDNA signal linked to 5S rDNA on chromosome 15 and one 45S rDNA signal unlinked to 5S rDNA on chromosome 17 in Bixiang seedless grape.【Conclusion】The optimal method for preparing metaphase chromosomes of grape root tips is pretreatment with APM for 2 h，and 1 MPa N₂O for 30 min，using the flamedrying method for slide preparation. The 45S rDNA and 5S rDNA are linked on grape chromosomes，with varying numbers in diploid，triploid，and tetraploid grapes. The 45S rDNA and 5S rDNA of 14 grape varieties exhibited an L-shape arrangement on the chromosomes，and the number of 5S rDNA loci is more stable than that of 45S rDNA loci.