Don't store the Graphic object. Just create it from the bitmap when you need to draw something:
Private Sub PictureBox1_MouseMove(sender As Object, e As MouseEventArgs) Handles PictureBox1.MouseMove
If dwn = True Then
Using g As Graphics = Graphics.FromImage(bit)
Dim s As Integer
Dim xy As Point
Using br2 As New SolidBrush(Color.FromName("White"))
s = Tloustka
br.Color = Color.FromName("White")
br.Width = Tloustka
xy.X = e.X
xy.Y = e.Y
If firstrun = True Then
lst = xy
firstrun = False
End If
g.FillEllipse(br2, xy.X - CLng(s / 2), xy.Y - CLng(s / 2), s, s)
g.DrawLine(br, lst, xy)
End Using
End Using
lst = xy
PictureBox1.Invalidate()
End If
End Sub
Then use the paint event of the PictureBox to show the image:
Private Sub PictureBox1_Paint(sender As Object, e As PaintEventArgs) Handles PictureBox1.Paint
e.Graphics.DrawImage(bit, Point.Empty)
End Sub
Make sure you dispose of your graphic objects.