Hevea brasiliensis is the sole commercial source of Natural Rubber. Development of haploids and homozygous diploids is of great significance in crop improvement. Conventional techniques for the production of homozygous plants are quite lengthy and tedious in the case of perennial tree crop like Hevea with a long breeding cycle. This paper describes optimization of protocols for the isolation and culture of embryo sacs for the development of gynogenic haploids. Three different methods were attempted for the isolation of embryo sacs, which were then cultures for callus induction in different media combinations. Embryo induction from the proliferated callus was tried tthrough evaluation of various parameters viz. basal media, growth regulators, phytagel and sucrose. Embryos were cultured in maturation media and later transferred to germination media. Pretreatment of ovule was identified as the ideal technique to isolate intact and viable embryo sacs. 36;callus induction was observed from cultured embryo sacs in modified K&M medium fortified with 4.6 mM2, 4-D and 2.7 mM Kinetin. Embryo induction frequency (30;) was obtained in K&M supplemented with GA3 (1.73 mM) and BA (7.3 mM) along with 8;sucrose and 0.5;phytagel. Mature embryos were obtained (8;) in modified MS medium containing IBA (0.74 mM) and Kinetin (2.3 mM) along ABA (1.89 mM). Mature embryos, upon transfer to plant regeneration medium, underwent biopolar differentiation and subsequently regenerated into planters at a very low frequency, with a low pace. Ploidy of the haploid callus was confirmed through cytological and flow cytometric analyses. This is the first report of development of gynogenic haploids, with confirmed ploidy, in Hevea brasiliensis.