Chikungunya virus (CHIKV) is an emerging arbovirus associated with large-scale epidemics, including a 2005-2006 epidemic on Reunion Island that resulted in approximately 266,000 human cases. The epidemic was associated with a strain of CHIKV carrying a mutation in the envelope protein gene (E1-A226V). This study investigated the impact of the E1-A226V mutation on CHIKV fitness in two mosquito species, Ae. aegypti and Ae. albopictus. The results showed that the E1-A226V mutation significantly increased CHIKV infectivity and dissemination in Ae. albopictus mosquitoes, leading to more efficient transmission to suckling mice. In contrast, the mutation had a marginal effect on infectivity in Ae. aegypti mosquitoes, no effect on viral dissemination, and a slight increase in transmission to suckling mice. The mutation also influenced CHIKV cholesterol dependence, suggesting a link between cholesterol dependence and increased fitness in Ae. albopictus. These findings provide insights into how a single amino acid substitution can influence vector specificity and epidemic potential, highlighting the vulnerability of urban areas in Europe and the United States to CHIKV establishment due to the widespread distribution of Ae. albopictus.Chikungunya virus (CHIKV) is an emerging arbovirus associated with large-scale epidemics, including a 2005-2006 epidemic on Reunion Island that resulted in approximately 266,000 human cases. The epidemic was associated with a strain of CHIKV carrying a mutation in the envelope protein gene (E1-A226V). This study investigated the impact of the E1-A226V mutation on CHIKV fitness in two mosquito species, Ae. aegypti and Ae. albopictus. The results showed that the E1-A226V mutation significantly increased CHIKV infectivity and dissemination in Ae. albopictus mosquitoes, leading to more efficient transmission to suckling mice. In contrast, the mutation had a marginal effect on infectivity in Ae. aegypti mosquitoes, no effect on viral dissemination, and a slight increase in transmission to suckling mice. The mutation also influenced CHIKV cholesterol dependence, suggesting a link between cholesterol dependence and increased fitness in Ae. albopictus. These findings provide insights into how a single amino acid substitution can influence vector specificity and epidemic potential, highlighting the vulnerability of urban areas in Europe and the United States to CHIKV establishment due to the widespread distribution of Ae. albopictus.