There are scarce information regarding Leptosphaeria maculans isolates from Portugal. This paper reports the first molecular study with Portuguese L. maculans isolates. PCR-ISSR and RAPD markers were used to study the genetic relationships of 18 Portuguese isolates of Leptosphaeria maculans from Beja region (south Portugal) to 12 other reference isolates representative of each group of the L. maculans species complex. Cluster and principal components analyses were conducted using PCR-ISSR and RAPD data from amplification with selected ISSR primers and RAPD markers, detecting 234 polymorphic fragments. The results showed that the 30 isolates clustered into two distinct groups (Tox(+) and Tox(0) isolates) and 4 subgroups: i) a large and compact subgroup containing all the Tox+ L. maculans "brassicae" isolates including all Portuguese isolates; ii) the unique "Lepidium" isolate; iii) a relatively heterogeneous subgroup with the Tox(0) NA2 L. biglobosa "canadensis" isolates; and iv) a dispersed subgroup with the other Tox(0) NA1 and NA3 L. biglobosa isolates and NA2 L. biglobosa "erysimii" isolate. There is low similarity between these three isolates. The Portuguese and foreign Tox(+) L. maculans "brassicae" isolates could be further divided into phenetic groupings/clusters. These groupings did not correspond to their pathogenicity groups revealed by plant differentials. The Portuguese L. maculans population do not seem to be different to that occurring in other countries (Australia, France, Germany, UK) which confirms that there is a large-scale mixing in L. maculans complex.