Abstract: The spiny eel (Mastacem belus aculeatus) is a species of the genusMastacembelus (Osteichthyes, Perciformes), whichmainly live in the freshwater of the south ofChina1 The fish isparticularly attractive for cytogenetical study ow ing to possessing well differentiated X and Y sex chromosomes.In the present study, the X chromosomesofM. aculeatus werem icrodissected from themetaphasesof chromosomes of the female. Then, the fragments of X chromosomeswere put into am icro tube and amplified using Degenerated Oligonucleotide-Primed PCR (DOP-PCR). Thereafter, the product of PCR was connected to T vector and the plasm idswere electric transformed into E1 coli1 As a resul,t the library of X chromosome ofM. aculeatus was constructed. The sum of the lengths of the inserted fragments is some 1.08 ?108bp, w ith an average length of 500bp. It is believed that the library coveredmore than 98% sequences of the whole X chromosome theoretically.To check the credibility of the library, fluorescence in situ hybridization (FISH) was applied. The fragmentsof the library were labeledw ith biotin by PCR. Then the probeswere hybridized to the chromosomem etaphases ofboth sex ofM 1 aculeatus in the absence or the presence of competitorDNA1 Strong signalswere detected on the entire sex chromosomes aswell as signals dispersed on all autosomesw ith the condition of FISH in the absence of competitor DNA.W hereas competitorDNA was added, the signals disappeared except those showed on heterochrom atic regions of X and Y chromosomes.H ence, we assorted the repetitive sequences in theX chromosom e ofM. aculeatus into three types. It was interpreted that the signalsof FISH in the presence of competitor DNA showed the distribution of typerepetitive sequences on the sex chromosomes ofM. aculeatus.