Eucalyptus cladocalyx F. Muell is a tree endemic to southern Australia and is distributed across four isolated regions: Kangaroo Island, southern Flinders Ranges, and two geographical zones in Eyre Peninsula. E. cladocalyx is capable of growing under extreme environmental conditions, including dry and saline soils. The objective of this study was to analyze genetic diversity in 45 half-sib families planted in northern Chile that are distributed across five different zones (provenances). Genetic variability was assessed using ISSR (Inter Simple Sequence Repeat) molecular markers. The results showed low levels of genetic diversity within populations (He = 0.113 to 0.268) in contrast with other Eucalyptus species. In addition, there was a significant genetic differentiation among provenances (Φst = 0.14); populations from the Kangaroo Island provenance showed more differentiation than any other population. These results are in agreement with previous studies of the species. Our study revealed that Chilean resources are a representative sample of Australian populations; therefore, the germplasm planted in northern Chile would be sufficient for the development of improvement programs. ISSR-Marker technology could be an alternative to identify genotypes of interest in material selection.