Most cases of nosocomial bacteremia are catheter related, and coagulase-negative staphylococci (CoNS) are the microorganisms most frequently associated with these infections. Subtle morphological differences are frequently found among CoNS colonies cultured from infected catheters. The aim of this study was to analyze the significance of the morphological heterogeneity observed in these CoNS populations. With this purpose in mind, the clonal composition of the CoNS populations obtained from a selection of nine catheters was analyzed by two different molecular techniques, arbitrarily primed-PCR and DNA macrorestriction analysis by pulsed-field gel electrophoresis. Twenty CoNS morphotypes were included for analysis, and four single colonies representative of each morphotype were selected. Morphological differences between colonies were found to correlate in all cases with differences at the molecular level. Unique fingerprints were also obtained for some isolates which were indistinguishable from other representatives of the same morphotypes. Differences in the molecular patterns among the isolates were associated in most of the cases with differences in the antimicrobial susceptibility patterns. The frequent isolation of polyclonal CoNS populations from catheters, with heterogeneous antimicrobial susceptibility patterns, has relevant epidemiologic and therapeutic implications in the context of catheter-related infections.