Despite the fact that lime and lemon groups have been collected as germplasm in the Indonesian Citrus and Subtropical Research Institute, a limited amount of research has been conducted on the identification and evaluation of these species. This study aimed to evaluate the morphological, anatomy, genetic polymorphism, and relationships among 16 lime and lemon accessions based on six ISSR markers. The research was conducted at Punten Experimental Garden, Batu, East Java, Indonesia from January to December 2021 in order to describe the morphology and anatomy characteristics. The results showed that 109 DNA bands were considered for the ISSR analysis, of which 88.1% were polymorphic. The dendrogram showed that the accessions were separated into two main groups at a coefficient of 0.62. The first group was divided into three clusters, i.e. A, B, and C consist of introduced varieties, most of the local lemons, and limes, respectively. The second group has only a single member (Limo Kwit). The closest genetic distances were between Cai Kahuripan and Lemon Jumbo (88.1%) and Uereka with Lime Bearr (88.3%). In contrast, the lowest ones were Montaji with Lemo Kwit (52.9%) and Lemon Swanggi with Jari Buda (51.2%). In general, genetic traits reflected in existing clusters contributed to differences in their morphology and anatomy of plants. It is assumed that the local accession of Lemo Kwit is a hybrid of subgenus papeda, so it is separated from the lime and lemon groups that belong to Cluster II. This finding is very useful for breeding programs in Indonesia.