Hannah, who only returned to racing in April after breaking her ankle in August 2015, outsprinted Alice in the final 100m, with the sisters stealing a march on Lucy Garner, who finished third.
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Dame Sarah Storey laid down one of the day’s best attacks, going solo from within the first 20km of the race, with her lead standing at around 90 seconds at one point.
A strong 12-rider bunch eventually caught the time trial bronze medallist, with Storey momentarily dropping off the bunch but clawing her way back on as the group reached the final circuits round Stockton.
Along with the Barnes sisters, the elite group contained Garner, Storey, Nikki Harris, Jennifer George, Lucy Shaw, Neah Evans, Molly Weaver, Claire Rose, Annasley Park and Dani King.
With two laps to go the peloton were slowly gaining on the lead group, but with 38 seconds to make up in the last 10km it looked likely the winner would come from the group up the road.
Hannah Barnes tried her hand from the front as the group led into the final lap, but her attack was quickly shut down by King and the others.
But as the peloton chased behind the front group were preparing for a bunch sprint. The Barnes duo were wheel to wheel in the final 100m, with Garner and King a bike length behind.
Not often do you see the second-place rider smiling in a situation like this, but Alice was beaming as her sister crossed the line ahead of her.
National Championships women’s road race, Stockton-on-Tees (106km)
1. Hannah Barnes (Canyon-SRAM)
2. Alice Barnes (Drops Cycling)
3. Lucy Garner (Wiggle-High5)
4. Dani King (Wiggle-High5)
5. Jennifer George (Drops Cycling)