Abstract Haploid production using in vitro ovule cultures has long been recognized as an important tool to produce haploid and homozygous double-haploid plants for genetic studies and plant breeding programs. In the present study, four experiments were carried out to study the influence of genotype, position of female flowers on plant stem, temperature and sucrose concentration on the in vitro gynogenesis induction of squash. (1) Ovules of 12 genotypes were excised from female flowers, 1 day before anthesis, and cultured onto MS medium containing 3% sucrose and 1 mg l −1 from each of kinetin and 2,4-D (2,4- dichlorophenoxy acetic acid). Differences in response among genotypes were demonstrated. Raad F 1 showed the highest percentage of responding ovules and number of plantlets per dish with 48.8% and 15 plants, respectively. The results revealed that genotype is a key factor influencing the in vitro gynogenesis in squash. (2) Ovules were excised from first, second and third female flower of two hybrids (Giad and Raad) and cultured onto the mentioned above medium. The highest percentage of responding ovules and number of plantlets per dish were obtained from ovules excised from the second female flower on the plant stem. (3) Effect of temperature (4 and 32 °C) for 0, 4, 7 and 12 days on the ovule culture of Queen F 1 was studied. Ovules incubated at 4 or 32 °C for 4 days produced a better embryogenic response. (4) Three sucrose concentrations (30, 60 and 90 g l −1 ) were tested with the ovule cultures of the local cultivar (Eskandrani). Differences among sucrose concentrations were statistically significant and ovules cultured on the MS medium containing 30 g l −1 produced the best result. MS medium containing 90 g l −1 did not produce gynogenic ovules. Cytological studies reveal that about 65% of the regenerants were haploid (2 n = x = 20 chromosomes) and 35% were double haploid (2 n = 2 x = 40 chromosomes).