To study the molecular epidemiology of Pseudomonas aeruginosa, 53 isolates from different anatomic areas from patients of the Complejo Metropolitano Armodio Arias Madrid de la Caja del Seguro Social, were obtained and analyzed. The isolates of P. aeruginosa were obtained during a period of 1 year and were phenotypically identified by biochemical analysis carried out with the API 20NE (Bio Merieux, Brussels, Belgium) and antibiotic sensitivity of the strains as well. Molecular diagnosis was performed using Multiplex-PCR of the pyoverdin receptor genes FPVA. This study showed low specificity of phenotypic tests in use since 39 isolates from a total of 53 could not be identified as P. aeruginosa by using the API 20E system, representing 74% of the strains tested. Differences in susceptibility to antibiotics were found compare to previous studies, especially major changes in the antimicrobial susceptibility profile of quinolones were observed. All isolates were identified with any of the pyoverdin receptor genes. The availability of sequences of the pyoverdin receptor fpvA genes of P. aeruginosa allowed us to standardize a rapid technique for the siderotyping of all the isolated strains. This is extremely important for strains that do not produce pyoverdin, which cannot be detected by conventional culture methods. Our study showed that this test has demonstrated to be specific and rapid, when it was compared with the API 20NE test and the conventional antimicrobial sensitivity tests.