The 2018 outbreak of myxomatosis in the Iberian hare (Lepus granatensis), has been hypothesized to originate from a species jump of the rabbit-associated myxoma virus (MYXV), after natural recombination with an unknown poxvirus. Iberian hares were long considered resistant to myxomatosis as no prior outbreaks were reported. To provide insights into the emergence of this recombinant virus (ha-MYXV), we investigated serum samples from 451 Iberian hares (88 live captured, 313 hunted and 50 found dead) collected over two time periods, 1994-1999 and 2017-2019, using a rabbit commercial indirect ELISA after validation, and tested different tissues or sera by a qPCR targeting M0005L/R gene conserved in MYXV and ha-MYXV. The cut-off of ELISA Relative Index 10 = 6.1 yielded an estimated positive predictive value of 96.4% (CI95% 82.6-98.0%), by comparison with qPCR positive and negative reference hares. Overall, antibodies were detected in 12.6% (57/451) of the hares tested, of which 40.3% (23/57) were also qPCR positive. Antibodies were found in apparently healthy hares sampled in 1994-1999 (n=10, none MYXV-DNA positive), and in 2017-2019 (n=28, of which 14% were MYXV-DNA positive). For the Iberian hares hunted or live trapped, seroprevalence was significantly higher in 2017-2019 (13.0%, CI95% 9.2-18.2%) than in 1994-1999 (5.4%, CI95% 3.0-9.6%) (p=0.005), and significantly higher in 2019 (p=0.007), being lower during the winter (p<0.001). While our molecular and serological results show that Iberian hares have been in contact with MYXV or an antigenically similar virus at least since 1994, they also show an increase in seroprevalence in 2018-2019. The more remote contact of hares with MYXV may have occurred with strains that circulated in wild rabbit, or unnoticed strains circulating in Iberian hare populations. This work clearly confirms the circulation of MYXV in the Iberian hare ate least 20 years before the severe virus outbreaks observed in 2018.