FLU (Friend leukemia virus integration 1), a member of the Ets gene family, is disrupted on llq24 by the Ewing’s Sarcoma (ES) and Peripheral Neuroepithelioma (PNE) t(11;22)(q24;q12) translocation. ES and PNE are Primitive Neuroectodermal Tumors (PNETs) and the consistent translocation t(11;22)(q24;q12) can be used for differential diagnosis. In PNETs the 3’ part of human FLU is translocated from 11q24 to 22q12, where it is juxtaposed to the 5’ end of the Ewing’s Sarcoma gene (EWS). A fusion transcript, resulting in a chimeric protein, is generated. Here, we present the isolation and detailed characterization of a 250-kb colinear YAC, B45C11, which encompasses the ES and PNE breakpoint on 11q24, as shown by FISSH on ES and PNE chromosomes and interphase nuclei. This YAC represents a new reagent for potential use in rapid differential diagnosis by FISSH on tumor biopsies and on paraffin embedded samples, particularly when DNA and/or RNA are not available for molecular analysis. YAC B45C11, which spans 250 kb of contiguous DNA around the ES and PNE breakpoint, contains the entire FLU gene. Three potential HpaII-tiny-fragment (HTF) islands are revealed within the YAC. One of these islands appears to be associated with the 5’ end of FLU, which extends over approximately 120 kb of DNA on 1 lq24. In addition, we demonstrate that YAC B45C11 contains other transcribed sequences in addition to FLU, by “cross-species” Northern blot hybridizations, which suggests the presence of additional genes in the immediate vicinity of the ES breakpoint on 1 lq24.