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Attempts to obtain new variants of roselle through conventional breeding were unsuccessful for 358 trials of (UKMR-3 × UKMR-2) and 63 trials of (UKMR-3 × accession 3). Roselle is an autotetraploid plant (2n = 4x = 72). Therefore the objectives of this study were to obtain new variants in roselle through the establishment of a plant regeneration system via microspore culture followed by detection of the ploidy level of regenerants based on their distinctive PCR patterns. The effects of different media and hormonal combinations were evaluated on microspore culture of roselle. This method was effective for callus induction, early-stage embryo development and subsequently efficient plant regeneration. Pretreatment of microspores at 4 °C and 35 °C for 3 days in the dark and without pretreatment, showed significant results on percentage of callus induction. Thirty regenerants were evaluated for their ploidy level using flow cytometry combined with propidium iodide and only 1 was detected as haploid, 4 mixploids and 25 diploids. Polymerase chain reaction (PCR) analysis using M13 universal primer (5′-TTATGAAACGACGGCCAGT-3′) showed the regenerated haploid plant having four unique bands at loci M13-01, M13-03, M13-04, and M13-06, which were absent in the microspore donor plant UKMR-1. To our knowledge, this is the first report of the development of haploid plants in roselle via microspore culture.