Route 27 begins as a water-logged route at its western terminus, requiring Trainers to surf east from New Bark Town to reach the first area of land along the route. After making landfall for the first time, a helpful Trainer near the shore will notify other Trainers that they have officially entered Kanto. The route continues east to a three-way intersection. The east path leads to the lower portion of a ledge and, ultimately, a dead end. The lack of a through route to the east forces Trainers to the north, where the path meets the western portal of the Tohjo Falls.
A Trainer awaits at the eastern portal, battling unsuspecting Trainers who exit the area. Also around this area is a house owned by a woman who can detect how devoted a player's Pokémon is to them. If a Trainer shows her that their lead Pokémon is attached to them, the woman will give them TM37 (Sandstorm). This prize is only attainable once regardless of how many different Pokémon are shown to her. South of the house is the aforementioned ledge used to bypass the Tohjo Falls when heading west along the route.
The route crosses a pond before resuming its course on land. This area of the route is home to the lone patch of grass along Route 27. The path of Route 27 follows the grass southeast, passing a Trainer amongst the grass, before arriving at a bridge. There are two Trainers near the starting point, with a third, Cooltrainer ReenaGSC / Ace Trainer ReenaHGSS, on a north-south segment east of the starting location. The beginning of this segment is also the western terminus of an express path that originates on Route 26.
After battling, Reena will offer to exchange numbers in the Pokégear for future rematches.
East of Reena, the bridge turns east once again, becoming Route 26 eight paces to the east.
There are two water paths, both of which are optional.
Directly south of the west entrance to the Tohjo Falls is a shoreline separating the land-bound route from a water path. The path on water turns east shortly after heading south from the shore, paralleling Route 27 to its terminus on a tiny patch of land containing a Rare Candy.
Like on Route 28 in Pokémon HeartGold and SoulSilver, the player is unable to fly to Johto from this route but can fly to both Kanto and Johto from Route 26 and the Indigo Plateau despite Route 27 being closer to Johto. In Generation II, the player can only fly to Johto from Route 26-27 and Indigo Plateau prior to boarding the S.S. Aqua the first time. After arriving in Vermilion City, the player cannot fly to Johto from anywhere in Kanto, which includes Routes 26 to 28 and the Indigo Plateau.