In the Yucatán Peninsula, no reproductive analyses have been conducted on clones of the Brachionus plicatilis species complex, a group of significant economic, ecological and evolutionary importance. Recent studies have identified 15 distinct genetic lineages. Therefore, in this study, our aim was to cultivate and characterise clones within this group to study reproductive isolation in the Yucatán Peninsula, specifically in the State of Quintana Roo. Specimens were collected in the southeast and northwest of Quintana Roo using a Wisconsin-type zooplankton net with a 54-μm mesh size. Zooplankton samples were isolated and cultured in a bioclimatic chamber at 25°C with a photoperiod of 12:12 h of light and dark. After isolation, the rotifers were fed 1×106 cells ml-1 of Nanochloropsis oculata. Subsequently, following two months of acclimatisation to laboratory conditions, we conducted taxonomic identification, created monoclonal cultures, performed morphometric characterisation, determined the population growth and resting eggs hatching percentage and performed cross-mating experiments. Twelve and eleven clones were obtained from the northwest and southeast parts of Quintana Roo, respectively. Differences were observed in the production of females, males and resting eggs, as well as in the percentage of resting eggs hatching. We determined that the southeast and northwest Quintana Roo had two different strains of rotifers due to reproductive isolation between the two regions. Therefore, we propose two populations for the coasts of Quintana Roo: Brachionus cf. ibericus "Cancún" strain and "Sian Ka'an" strain.