Two multidrug-resistant Pseudomonas aeruginosa strains harboring blaVIM were isolated from a hospital in Egypt and were indistinguishable by pulsed-field gel electrophoresis. Sequence analysis revealed a novel VIM β-lactamase, VIM-28. Additional sequencing of integron revealed that the blaVIM-28 gene cassette had an unusual arrangement of class 1 integron structure, located directly downstream of the integrase gene "intI1" and oriented divergently from it. The new organization of integron also comprised the aacA7 and smr-2 gene cassettes in that order. A complete ISPa21 containing a tnpA gene flanked by two 13-bp inverted repeats was located directly upstream of the 3'-CS conserved region of the integron containing qacEΔ1/sul1 genes. The blaVIM-28-containing class 1 integron was found to have a chromosomal origin in both strains. In conclusion, the presence of a new variant of blaVIM, blaVIM-28, on a new organization of class 1 integron having ISPa21 increases the possibility of dissemination of resistance genes within the integron in P. aeruginosa among hospitalized patients in Egypt.