Happy Pride! Here is all canonically queer jrwi characters :))

Player Characters:

Gillion Tidestrider - Asexual

Jay Ferin - Bisexual

William Wisp - Bisexual

Rumi - Queer, unlabelled

Peter Sqloint - Queer (exact sexuality not confirmed)

In episode 11 of Apotheosis, Peter was revealed to have feelings for Rumi.

Kian Stone - Bisexual

Shilo Bathroy - Aromantic

In episode 7 of the Suckening, Bizly responded "he's not really capable of that" to Grizzly asking if Shilo has romantic feelings towards Edward Twilight after drinking his blood.

Br'aad Vengolor - Gay

Taxi - Pansexual

Kasper - Gay

Honorable mentions:

Vyncent Sol is implied to be romantically interested in William Wisp, who is canonically bisexual. It's unclear whether or not it's just a running joke, or genuine subtext. Additionally, in the same stream in which Condi confirmed Jay to be bisexual, he also mentioned Vyncent "doesn't have a canon sexuality".
Arthur Morgan developed some feelings for Deacon Keller after the blood exchange between them. However, since it was a result of vampire magic, it's hard to tell whether any genuine attraction was involved.
Velrisa Grayrock was implied to be bisexual by her player, Wheatie, in one of his streams, around a year after the Fated campaign was cut short.
Charlie once jokingly said Br'aad has a vagina when someone in the (now deleted) JRWI discord server compared the kazoo strapped to Br'aad's side to a tampon. This was most likely a joke, but it could imply Br'aad to be a trans man.

NPCs:

Lizzie - Lesbian

In episode 101 of Riptide, Lizzie has been confirmed to have dated a woman in the past. In addition to this, the beads in her hair in her official design are the colors of the lesbian flag.

Ezme - Genderfluid

Jasmine Drake - Trans man

(also there is canonically no transphobia in the Riptide world!)

Le frog - Queer (implied mlm)

Le Frog and Pencil Man being together has been a common joke between PD fans for a while, Bizly acknowledged it a few times as well and seemed to approve.

Honorable mentions:

Queen uses any pronouns.
The God of Charisma uses he/they pronouns.
Pencil Man is implied to have been romantically involved with Le Frog before the events of the Prime Defenders campaign.
Deacon Keller has developed some feelings for Arthur after the blood exchange between them. However, since it was a result of vampire magic, it's hard to tell whether any genuine attraction was involved.
Strangle MaCock was implied to reciprocate Kasper's feelings.