It was the first time the married couple jointly took part in the traditional procession along the course as they travelled with the Queen's first cousin, Princess Alexandra, and the Countess Mountbatten of Burma, Penelope Knatchbull.
WATCH: The royal family attend day two of Royal Ascot
However, royal watchers were quick to notice Princess Beatrice's sweet gesture towards her campions by travelling backwards.
It's believed that the Queen and members of the royal family tend to take the forward-facing seats since they have a better view of the crowds and the racecourse as they enter the enclosure. They also tend to be photographed more clearly.
On this occasion, Beatrice and Edoardo bent the rules and took the backward-facing seats - no doubt as a sign of respect to the Queen's cousin and close friend.
Princess Beatrice and Edoardo took the backward-facing seats in the carriage
They joined the procession with Prince Charles and the Duchess of Cornwall, who were sat in the first carriage with Vice Admiral Sir Tony Johnstone-Burt, leaving an empty seat in honour of the Queen's absence.
Her Majesty has been forced to miss the sporting event she is passionate about, but is likely to have watched proceedings on television from Windsor Castle and been kept abreast of developments by her racing advisor John Warren.
They were joined by Princess Alexandra and the Countess Mountbatten of Burma
The Queen has more than ten horses entered over the five days of the event and, with her Berkshire home just a 15-minute drive away, there is still some hope she will attend during the week.
Prince Edward and the Countess of Wessex were also in attendance alongside the likes of the Queen's cousin Flora Vesterberg.
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