Lessonia nigrescens and L. trabeculata are economically important kelp species in northern Chile. Despite their considerable value, the genetic diversity of these kelp has received limited attention in the Atacama region. This study aimed to analyze genetic diversity, establish genetic relationships, and identify diagnostic DNA markers in the Atacama region's L. nigrescens and L. trabeculata. Both species' Inter-sequence simple repeat (ISSR) band patterns showed high reproducibility and reliability. Among 17 ISSRs, 161 polymorphic bands were identified in Lessonia spp. Parameters, such as percentage polymorphism, number of alleles per locus, effective number of alleles, and Shannon information index, indicated that L. nigrescens had slightly higher genetic diversity than L. trabeculata. The Unweighted pair group method with arithmetic average (UPGMA) cluster analysis revealed two main clusters: one containing L. nigrescens and the other containing L. trabeculata. Additionally, four species diagnostic ISSR markers were identified for L. nigrescens and L. trabeculata. The ISSR markers proved valuable in detecting genetic diversity in L. nigrescens and L. trabeculata. This information is particularly important for marine management strategies and ensuring successful conservation.