The newly developed technique of amplified fragment length polymorphism (AFLP) was used to study the polymorphism and segregation of AFLP markers in an interspecific cross of oil palm. The technique revealed a high level of polymorphism between the Colombian oleifera (female parent) and Nigerian guineensis (male parent) used to generate the cross. The segregation of AFLP markers was studied in a population of 77 F, individuals and analysed as a pseudo-testcross. Using only 10 primer pair combinations, we were able to amplify 674 bands out of which 91 show segregation. All markers detected were scored as dominant, and the segregation ratios indicated that majority of these markers (80%) were inherited in a Mendelian manner. The results also showed that the male parent (palm T128, a Nigerian guineensis) was more heterozygous than the female parent (UP1026, a Colombian oleifera). The AFLP technique was also found to be suitable for detecting contaminants and would therefore be useful for assessing the fidelity of controlled crosses.