In 1998, the Colombian government initiated an immunization program for children under one year of age with a Haemophilus influenzae capsular type b conjugate vaccine. After two years, the surveillance program of the Colombian Instituto Nacional de Salud Microbiology Group reported a 40% decrease in meningitis cases caused by H. influenzae. This effect was attributed to the vaccination. The surveillance program uses the standardized slide agglutination technique to serotype H. influenzae. The current study validated the accuracy of the slide agglutination method by means of the PCR technique. From children under five years of age, 146 isolates were obtained. These were collected between 1999 and 2002, and were characterized by biochemical tests and serotyped by the INS as part of the surveillance program. PCR confirmed 93% of the H. influenzae serotype b and 92% of the other serotypes. When the slide agglutination technique is conducted under a strict quality control program, it remains a sensitive and specific tool for serotyping H. influenzae.