Pineapple production and productivity can increased by the use of superior variety. Pertaining to variety assembling, the relationship information among parents are needed to gain heterosis effect through molecular identification activity. This research was aimed to (1) determine the level of polymorphism primers used, (2) identify specific DNA fragments which discrete individual or group of pineapple, and (3) reveal genetic relationship among pineapple species and accessions. The experiment was conducted on May to December 2014 in Seeds Quality Testing and Molecular Laboratory of Indonesian Tropical Fruit Research Institute. Nineteen accessions from four species (Ananas comosus, A. brachteatus, A. lucidus, and A. nanus) of pineapple were used as samples. Twenty rapid amplified polymorphism DNA (RAPD) markers were used on molecular analysis. The data were scored binary and then they were analyzed using NTSYSpc 2.1x computer software. The analysis showed that the 20 primers had 33–100% polymorphic with 87% in average. Primers with 100% polymorphism level were RAPD3, OPA13, OPAV3, OPC12, OPC16, and OPY15. Cayenne group could be denoted with RAPD1 and OPAV3 markers by 1,000 base-pairs (bp) and 700 bp band, respectively. Meanwhile the Queen group can be identified by 700 bp band RAPD3 marker. The Spanish group can be specified by1,500 bp band RAPD2 and RAPD3 markers. Based on cluster analysis the 19 accessions were separated into six groups with 0.75 genetic similarity coefficient i.e., Queen, Cayenne, Spanish, A. bracteatus, A. lucidus, and A. nanus. These accessions had a wide genetic diversity with 0.41 to (0.85) genetic similarity coefficients. The highest genetic similarity coefficient (0.85) was determined between N-73 and BB, whereas the lowest value down to 0.41 was indicated on N-94 (A. nanus) and N-18 (Green Spanish). The implications of this research are that one of two accessions that have high genetic similarities can be eliminated for efficiency in the management of germplasm. While accessions which have little genetic similarity are both used as crosses parent in order to obtain wide genetic variability and high heterosis effects.