To determine pseudopterosin composition and concentration in colonies of Pseudopterogorgia elisabethae from the islands of San Andres and Providencia, we collected fragments of individual colonies at various sites and depth ranges around the islands. Chromatographic profiles of the polar fraction, particularly those obtained by HPLC-MS analyses, allowed us to recognize two different chemotypes. Chemotype 1 characterized samples from Providencia whereas chemotype 2 characterized samples from San Andres. A complex pseudopterosin mixture (compounds 1-13) characterized chemotype 1. These compounds were isolated by a combination of chromatographic methods and identified by spectroscopic methods (MS, UV, 1H, and 13C NMR). We identified the known pseudopterosins G and K and seco-pseudopterosin A. We also isolated and identified seven new compounds, pseudopterosins P-V, isomers of known pseudopterosins. Pseudopterosins G and K were found at concentrations ranging between 1 and 3% of the animal dry mass. Pseudopterosins Q and U were the major compounds reaching up to 6% of the animal dry mass at some locations. Major metabolites in chemotype 2 had a molecular weight and fragmentation pattern different from that observed in the pseudopterosins, as determined by HPLC-MS. Total pseudopterosin concentration in this chemotype was below 3% dry mass at all sites. Total pseudopterosin concentration was significantly higher in chemotype 1, with concentrations ranging between 4 and 20% dry mass. At most locations on Providencia, however, total pseudopterosin concentration ranged between 11 and 15% dry mass. Concentrations exceed reports from other locations in the Caribbean. Furthermore, pseudopterosin composition in our samples is quite different from those in specimens of P. elisabethae from the Bahamas and Bermuda. Pseudopterosins G, K, and P-V are characteristic of P. elisabethae colonies from the island of Providencia, while pseudopterosins A-D are characteristic of colonies of P. elisabethae from the Bahamas islands, and pseudopterosins E-L have been isolated from P. elisabethae from the Bahamas and Bermuda. The overall morphology of P. elisabethae can be variable, and chemical differences are not correlated to specific morphs. We confirmed the species identity of each colony by morphological and sclerite analysis and found no significant differences in sclerite dimensions among different colonies and chemotypes.