Introduction: Insertion sequences such as ISCR1 promote capture, transposition and expression of blaCTX-M genes. Thus, gene dissemination in bacterial populations occurs rapidly.Objective: To determine the presence of ISCR1 sequence genes and their association with blaCTX-M-1 and blaCTX-M-2 on plasmids IncN and IncFIIA from K. pneumoniae of nosocomial origin, was determined.Materials and methods: Three strains of K. pneumoniae with reduced susceptibility to extended-spectrum cephalosporins were isolated from neonatal sepsis cases of nosocomial origin. Phenotypictests showed the presence of ESBLs. Plasmids were isolated and classified according to incompatibility groups by PCR replicon typing. Detection and association of ISCR1 with blaCTX-M genes were determined by PCR and direct sequencing through the use of several sets of PCR primers.Results: All strains showed phenotypic profile consistent with ESBL-producing transferred byconjugation. PCR amplification assay for CTX-M together with sequencing analysis revealed that strains carrying blaCTX-M-1 y blaCTX-M-2 genes were localized in plasmids of approximately 150 kb relatedto IncN and IncFIIA groups, respectively. ISCR1 was found upstream and associated with blaCTX-M-1 yblaCTX-M-2 genes.Conclusion. Thus far, this is the first Venezuelan report, in which ISCR1 presence is closely relatedto blaCTX-M-1 y blaCTX-M-2 gene mobilization in IncN and IncFIIA conjugative plasmids located in K.pneumoniae strains circulating at a neonatal high risk unit. doi: http://dx.doi.org/10.7705/biomedica.v33i2.774